Fused bicyclic 2,4-diaminopyrimidine derivative as a dual alk and fak inhibitor

ABSTRACT

The present invention provides a compound of formula (I) 
     
       
         
         
             
             
         
       
     
     or a salt form thereof. The compound of formula (I) has ALK and FAK inhibitory activity, and may be used to treat proliferative disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/824,750, filed Aug. 12, 2015, which is a continuation of U.S.application Ser. No. 14/474,928, filed Sep. 2, 2014, now U.S. Pat. No.9,132,128 (issue date Sep. 15, 2015), which is a continuation ofInternational Application No. PCT/US2013/029304, filed Mar. 6, 2013,which claims the benefit of U.S.

Provisional Application No. 61/607,305, filed Mar. 6, 2012, thedisclosures of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

Anaplastic Lymphoma Kinase (ALK) is a cell membrane-spanning receptortyrosine kinase, which belongs to the insulin receptor subfamily. Themost abundant expression of ALK occurs in the neonatal brain, suggestinga possible role for ALK in brain development (Duyster, J. et al.,Oncogene, 2001, 20, 5623-5637).

ALK is also implicated in the progression of certain tumors. Forexample, approximately sixty percent of anaplastic large cell lymphomas(ALCL) are associated with a chromosome mutation that generates a fusionprotein consisting of nucleophosmin (NPM) and the intracellular domainof ALK. (Armitage, J. O. et al., Cancer: Principle and Practice ofOncology, 6^(th) edition, 2001, 2256-2316; Kutok J. L. & Aster J. C., J.Clin. Oncol., 2002, 20, 3691-3702). This mutant protein, NPM-ALK,possesses a constitutively active tyrosine kinase domain that isresponsible for its oncogenic property through activation of downstreameffectors. (Falini, B. et al., Blood, 1999, 94, 3509-3515; Morris, S. W.et al., Brit. J. Haematol., 2001, 113, 275-295; Duyster et al.; Kutok &Aster). In addition, the transforming EML4-ALK fusion gene has beenidentified in non-small-cell lung cancer (NSCLC) patients (Soda, M., etal., Nature, 2007, 448, 561-566) and represents another in a list of ALKfusion proteins that are promising targets for ALK inhibitor therapy.Experimental data have demonstrated that the aberrant expression ofconstitutively active ALK is directly implicated in the pathogenesis ofALCL and that inhibition of ALK can markedly impair the growth of ALK+lymphoma cells (Kuefer, Mu et al. Blood, 1997, 90, 2901-2910; Bai, R. Y.et al., Mol. Cell Biol., 1998, 18, 6951-6961; Bai, R. Y. et al., Blood,2000, 96, 4319-4327; Ergin, M. et al., Exp. Hematol., 2001, 29,1082-1090; Slupianek, A. et al., Cancer Res., 2001, 61, 2194-2199;Turturro, F. et al., Clin. Cancer Res., 2002, 8, 240-245). Theconstitutively activated chimeric ALK has also been demonstrated inabout 60% of inflammatory myofibroblastic tumors (IMTs), a slow-growingsarcoma that mainly affects children and young adults. (Lawrence, B. etal., Am. J. Pathol., 2000, 157, 377-384; Duyster et al.).

In addition, ALK and its putative ligand, pleiotrophin, areoverexpressed in human glioblastomas (Stoica, G. et al., J. Biol. Chem.,2001, 276, 16772-16779). In mouse studies, depletion of ALK reducedglioblastoma tumor growth and prolonged animal survival (Powers, C. etal., J. Biol. Chem., 2002, 277, 14153-14158; Mentlein, R. et al, J.Neurochem., 2002, 83, 747-753).

An ALK inhibitor would be expected to either permit durable cures whencombined with current chemotherapy for ALCL, IMT, proliferativedisorders, glioblastoma and possible other solid tumors, or, as a singletherapeutic agent, could be used in a maintenance role to prevent cancerrecurrence in those patients. Various ALK inhibitors have been reported,such as indazoloisoquinolines (WO 2005/009389), thiazole amides andoxazole amides (WO 2005/097765), pyrrolopyrimidines (WO 2005080393), andpyrimidinediamines (WO 2005/016894).

WO 2008/051547 discloses fused bicyclic derivatives of2,4-diaminopyrimidine as ALK and c-Met inhibitors. The lead drugcandidate disclosed in the '547 application is CEP-28122, a potent ALKinhibitor with oral efficacy against SUP-M2 and Karpas-299 ALK-dependenttumors in mouse xenograft models. CEP-28122 progressed to IND-enablingstudies until its development was terminated due to the unexpectedoccurrence of severe lung toxicity in CEP-28122-treated monkeys.

Focal adhesion kinase (FAK) is an evolutionarily conserved non-receptortyrosine kinase localized at focal adhesions, sites of cellular contactwith the ECM (extra-cellular matrix) that functions as a criticaltransducer of signaling from integrin receptors and multiple receptortyrosine kinases, including EGF-R, HER2, IGF-R1, PDGF-R and VEGF-R2 andTIE-2 (Parsons, J T; Slack-Davis, J; Tilghman, R; Roberts, W G. Focaladhesion kinase: targeting adhesion signaling pathways for therapeuticintervention. Clin. Cancer Res., 2008, 14, 627-632; Kyu-Ho Han, E;McGonigal, T. Role of focal adhesion kinase in human cancer—a potentialtarget for drug discovery. Anti-cancer Agents Med. Chem., 2007, 7,681-684). The integrin-activated FAK forms a binary complex with Srcwhich can phosphorylate other substrates and trigger multiple signalingpathways. Given the central role of FAK binding and phosphorylation inmediating signal transduction with multiple SH2- and SH3-domain effectorproteins (Mitra, S K; Hanson, D A; Schlaeper, D D. Focal adhesionkinase: in command and control of cell motility. Nature Rev. Mol. CellBiol., 2005, 6, 56-68), activated FAK plays a central role in mediatingcell adhesion, migration, morphogenesis, proliferation and survival innormal and malignant cells (Mitra et al. 2005; McLean, G W; Carragher, NO; Avizzienyte, E; et al. The role of focal adhesion kinase in cancer—anew therapeutic opportunity. Nature Reviews Cancer, 2005, 5, 505-515;and Kyu-Ho Han and McGonigal, 2007). In tumors, FAK activation mediatesanchorage-independent cell survival, one of the hallmarks of cancercells. Moreover, FAK over expression and activation appear to beassociated with an enhanced invasive and metastatic phenotype and tumorangiogenesis in these malignancies (Owens, L V; Xu, L; Craven, R J; etal. Over expression of the focal adhesion kinase (p125 FAK) in invasivehuman tumors. Cancer Res., 1995, 55, 2752-2755; Tremblay, L; Hauck, W.Focal adhesion kinase (pp125FAK) expression, activation and associationwith paxillin and p50CSK in human metastatic prostate carcinoma. Int. J.Cancer, 1996, 68, 164-171; Kornberg, U. Focal adhesion kinase in oralcancers. Head and Neck, 1998, 20: 634-639; Mc Clean et al 2005; Kyu-HoHan and McGonigal, 2007) and correlated with poor prognosis and shortermetastasis-free survival.

Multiple proof-of-concept studies conducted in various solid tumorsusing siRNA (Halder, J; Kamat, A A; Landen, C N; et al. Focal adhesionkinase targeting using in vivo short interfering RNA delivery in neutralliposomes for ovarian carcinoma therapy. Clin. Cancer Res., 2006, 12,4916-4924), dominant-negative FAK, and small molecule FAK inhibitors(Halder, J; Lin, Y G; Merritt, W M; et al. Therapeutic efficacy of anovel focal adhesion kinase inhibitor, TAE226 in ovarian carcinoma.Cancer Res., 2007, 67, 10976-10983; Roberts, W G; Ung, E; Whalen, P; etal. Anti-tumor activity and pharmacology of a selective focal adhesionkinase inhibitor, PF-562,271. Cancer Res., 2008, 68, 1935-1944; Bagi CM; Roberts G W; and Andersen C J. Dual focal adhesion kinse/Pyk2inhibitor has positive effects on bone tumors—implications for bonemetastases. Cancer, 2008, 112, 2313-2321) have provided pre-clinicalsupport for the therapeutic utility of FAK inhibition as ananti-tumor/anti-angiogenic strategy, particularly forandrogen-independent prostate cancers, breast cancers, and HNSCCs. Inpreclinical models of human breast cancer (MDA-MB-231) in nude rats,administration of a small molecule FAK inhibitor (PF-562,271) inhibitedprimary tumor growth and intra-tibial tumor spread, and restoredtumor-induced bone loss (Bagi et al., 2008). Roberts et al., (2008)showed that PF-562,271 inhibited bone metastases, prevented boneresorption, and increased osteogenesis in breast andandrogen-independent prostate cancer patients with and without bonemetastases, supporting an additional benefit of FAK inhibition in thesespecific malignancies.

In summary, there is clear genetic and biological evidence that linksaberrant ALK activation and constitutive activation of FAK with theonset and progression of certain types of cancer in humans. Considerableevidence indicates that ALK- and FAK-positive tumor cells require theseoncogenes to proliferate and survive, and in the case of FAK, to invadeand metastasize to distant sites, while inhibition of both ALK and FAKsignaling leads to tumor cell growth arrest or apoptosis, resulting inobjective cytoreductive effects. Inhibition of FAK also results inattenuation of tumor motility, invasiveness, and metastatic spread,particularly in specific cancers characterized by bone metastaticdissemination and osteolytic disease. FAK activation protects tumorcells from chemotherapy-induced apoptosis, contributing to tumorresistance; modulation of FAK activity (by siRNA or pharmacologically)potentiates efficacy of chemotherapeutic agents in vivo (e.g.,doxorubicin, docetaxel and gemcitabine), suggesting the utility forrational combination therapies in specific cancers. ALK and FAK areminimally expressed in most normal tissues in the healthy adult and areactivated and/or dysregulated in specific cancers during oncogenesisand/or during early stages of malignant progression. Consequently, theon-target effects of treatment with a dual ALK and FAK inhibitor againstnormal cells should be minimal, creating a favorable therapeutic index.

A need exists for additional safe and effective ALK and/or FAKinhibitors for the treatment of cancer.

SUMMARY OF THE INVENTION

The present invention provides a compound of formula (I)

or a salt form thereof.

The compound of formula (I) has ALK and FAK inhibitory activity, and maybe used to treat ALK- or FAK-mediated disorders or conditions.

The present invention further provides a pharmaceutical compositioncomprising at least one compound of the present invention together withat least one pharmaceutically acceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an schematic overview of a process for preparingCEP-37440.

FIG. 2 depicts an XRPD pattern of a crystalline CEP-37440tribenzenesulfonate salt.

FIG. 3 depicts an XRPD pattern of a crystalline CEP-37440trihydrochloride dihydrate salt.

FIG. 4 depicts the anti-tumor efficacy of oral CEP-37440 in Sup-M2 ALCLtumor xenografts in mice.

FIG. 5 depicts the body weights of mice bearing Sup-M2 ALCL tumorxenografts dosed orally with CEP-37440.

FIG. 6 depicts the plasma and tumor levels of CEP-37440 in mice bearingSup-M2 ALCL tumor xenografts after oral dosing.

FIG. 7 depicts the anti-tumor efficacy of oral CEP-37440 in Karpas-299tumor xenografts in mice.

FIG. 8 depicts the body weights of mice bearing Karpas-299 tumorxenografts dosed orally with CEP-37440.

FIG. 9 depicts the plasma and tumor levels of CEP-37440 in mice bearingKarpas-299 tumor xenografts after oral dosing.

FIG. 10 depicts the anti-tumor efficacy of oral CEP-37440 in NCI-H2228NSCL tumor xenografts in mice.

FIG. 11 depicts the anti-tumor efficacy of oral CEP-37440 in NCI-H3122NSCL tumor xenografts in mice.

FIG. 12 depicts the plasma and tumor levels of CEP-37440 in mice bearingNCI-H2228 NSCL tumor xenografts after oral dosing.

FIG. 13 depicts the plasma and tumor levels of CEP-37440 in mice bearingNCI-H3122 NSCL tumor xenografts after oral dosing.

FIG. 14 depicts the body weights of mice bearing NCI-H2228 NSCL tumorxenografts dosed orally with CEP-37440.

FIG. 15 depicts the body weights of mice bearing NCI-H3122 NSCL tumorxenografts dosed orally with CEP-37440.

FIG. 16 depicts the anti-tumor efficacy and long term tumor regressionsof oral CEP-37440 in NCI-H2228 NSCL tumor xenografts in mice.

FIG. 17 depicts the body weights of mice bearing NCI-H2228 NSCL tumorxenografts dosed orally with CEP-37440.

FIG. 18 depicts the anti-tumor efficacy of oral CEP-37440 and PF-562271in nude mice bearing PC-3 prostate tumor xenografts with constitutiveFAK activation.

FIG. 19 depicts the anti-tumor efficacy of oral CEP-37440 and PF-562271in nude mice bearing HCC-827 human NSCL carcinoma xenografts (EML4-ALKnegative).

FIG. 20 depicts the body weights of nude mice bearing HCC-827 human NSCLcarcinoma xenografts (EML4-ALK negative) dosed orally with CEP-37440 orPF-562271.

FIG. 21 depicts the tumor pharmacodynamic inhibition of FAK andanti-tumor efficacy of oral CEP-37440 and PF-562271 in SCID mice bearingDetroit 562 human HNSCC carcinoma xenografts (EML4-ALK negative).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

“Pharmaceutical composition” refers to a composition having asafety/efficacy profile suitable for administration to a human.

“Pharmaceutically acceptable excipient” refers to physiologicallytolerable materials, which do not typically produce an allergic or otheruntoward reaction, such as gastric upset, dizziness and the like, whenadministered to a human.

“Pharmaceutically acceptable salt” refers to a salt having asafety/efficacy profile suitable for administration to a human.

“Subject” refers to a member of the class Mammalia. Examples of mammalsinclude, without limitation, humans, primates, chimpanzees, rodents,mice, rats, rabbits, horses, livestock, dogs, cats, sheep, and cows.

“Therapeutically effective amount” refers to an amount of a compoundsufficient to improve or inhibit worsening of symptoms associated with adisorder or condition being treated in a particular subject or subjectpopulation. It should be appreciated that determination of proper dosageforms, dosage amounts, and routes of administration is within the levelof ordinary skill in the pharmaceutical and medical arts.

“Treatment” refers to the acute or prophylactic diminishment oralleviation of at least one symptom or characteristic associated orcaused by a disorder being treated. For example, treatment can includediminishment of a symptom of a disorder or complete eradication of adisorder.

II. Compound

The present invention provides a compound of formula (I)

or a salt form thereof. The compound of formula (I) has the chemicalname2-[[5-chloro-2-[[(6S)-6-[4-(2-hydroxyethyl)piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl]amino]pyrimidin-4-yl]amino]-N-methyl-benzamide,and is also known as CEP-37440. We have discovered that the compound offormula (I) possesses surprising and unexpected properties in comparisonto CEP-28122 and other related compounds.

The salt form of the compound of formula (I) is preferablypharmaceutically acceptable. Pharmaceutically acceptable acid salt formsof the compound of formula (I) include, but are not limited to, saltsderived from inorganic acids such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydroiodic, phosphorous, and mixtures thereof, aswell as the salts derived from organic acids, such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic andaromatic sulfonic acids. Such acid salts thus include, but are notlimited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite,nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, caprylate, isobutyrate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, mandelate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and mixtures thereof. In certainembodiments, the acid salt is chosen from benzenesulfonate and chloride.In certain embodiments the acid salt is a chloride. In certainembodiments, the acid salt is a tribenzenesulfonate. In certainembodiments, the tribenzensulfonate salt is characterized by a XRPDpattern having one or more peaks selected from 7.62, 13.11, 13.76, and14.05±0.2 degrees 2θ. In certain embodiments, the tribenzensulfonatesalt is characterized by a XRPD pattern having one or more peaksselected from 6.85, 7.62, 8.01, 13.11, 13.76, 14.05, and 14.60±0.2degrees 2θ. In certain embodiments, the tribenzensulfonate salt ischaracterized by a XRPD pattern having one or more peaks selected from7.62, 13.11, 13.76, 14.05, 17.10, 17.86, and 18.10±0.2 degrees 2θ. Incertain embodiments, the acid salt is a trihydrochloride. In certainembodiments, the acid salt is a trihydrochloride dihydrate. In certainembodiments, the trihydrochloride dihydrate salt is characterized by aXRPD pattern having one or more peaks selected from 5.42, 8.86, 14.06,17.52 and 18.51±0.2 degrees 2θ. In certain embodiments, thetrihydrochloride dihydrate salt is characterized by a XRPD patternhaving one or more peaks selected from 5.42, 5.91, 8.86, 10.80, 11.79,14.06, 14.72, 17.02, 17.52 and 18.51±0.2 degrees 2θ. In certainembodiments, the acid salt is a trihydrochloride monohydrate.

The acid addition salts may be prepared by contacting the compound offormula (I) with a sufficient amount of the desired acid to produce thesalt in the conventional manner. The free base form of the compound offormula (I) may be regenerated by contacting the salt form with a baseand isolating the free base in the conventional manner.

The present invention includes the compound of formula (I) and saltforms thereof in any physical form, including amorphous or crystallinesolids in any polymorphic form, in any state of purity. Crystallinepolymorphic forms include unsolvated forms as well as solvated forms,such as hydrated forms. Methods of preparing crystalline and amorphousforms of organic compounds such as the compound of formula (I) are wellknown to those of ordinary skill in the art.

III. Pharmaceutical Composition

The present invention further provides pharmaceutical compositionscomprising a compound of the present invention (e.g., a compound offormula (I) or a pharmaceutically acceptable salt thereof), togetherwith a pharmaceutically acceptable excipient. For preparing apharmaceutical composition from a compound of the present invention,pharmaceutically acceptable excipients can be either solid or liquid. Anexcipient can be one or more substances which may act as, e.g., acarrier, diluent, flavoring agent, binder, preservative, tabletdisintegrating agent, or an encapsulating material. The pharmaceuticalcomposition may contain two or more compounds of the present invention(e.g., two or more different salt forms of the compound of formula (I)).Preferably, the pharmaceutical composition contains a therapeuticallyeffective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt form thereof. In one embodiment, the compositioncontains an amount of a compound of formula (I) or a pharmaceuticallyacceptable salt form thereof effective to treat an ALK- or FAK-mediateddisorder or condition. Preferably, a pharmaceutical composition of thepresent invention will cause a decrease in symptoms or disease indiciaassociated with an ALK- or FAK-mediated disorder as measuredquantitatively or qualitatively. The composition may also contain, inaddition to a compound of formula (I) or a pharmaceutically acceptablesalt form thereof and a pharmaceutically acceptable excipient, anothertherapeutic compound, such as a compound useful in the treatment ofcancer.

A compound of the present invention can be formulated as apharmaceutical composition in any form, such as a syrup, an elixir, asuspension, a powder, a granule, a tablet, a capsule, a lozenge, atroche, an aqueous solution, a cream, an ointment, a lotion, a gel, anemulsion, etc. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. Preferably,the pharmaceutical composition is a tablet or capsule. In oneembodiment, the pharmaceutical composition is a tablet. In anotherembodiment, the pharmaceutical composition is a capsule.

In powders, the excipient may be a finely divided solid in a mixturewith a finely divided active component. In tablets, the active componentmay be mixed with an excipient having the necessary binding propertiesin suitable proportions and compacted in the shape and size desired.Suitable excipients include magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoabutter, and the like.

The pharmaceutical composition preferably contains from 1% to 95% (w/w)of the active compound. More preferably, the pharmaceutical compositioncontains from 5% to 70% (w/w) of the active compound.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, may be melted and the activecomponent dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture may then be poured into convenient sized molds,allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions.Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and nonaqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. In the practice of this invention,compositions can be administered, for example, by intravenous infusion,orally, topically, intraperitoneally, intravesically or intrathecally.The formulations of compounds can be presented in unit-dose ormulti-dose sealed containers, such as ampoules and vials. Injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

A compound of the present invention, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Pharmaceutically acceptable excipients are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention (see, e.g., Remington: The Science and Practice ofPharmacy, 20th ed., Gennaro et al. Eds., Lippincott Williams andWilkins, 2000).

The quantity of active component in a pharmaceutical composition may bevaried or adjusted from, e.g., 1 mg to 1,000 mg, 5 mg to 500 mg, 10 mgto 300 mg, or 25 mg to 250 mg, according to the particular application.

The dose administered to a subject is preferably sufficient to effect abeneficial therapeutic response in the subject over time. The beneficialdose can vary from subject to subject depending upon, e.g., thesubject's condition, body weight, surface area, and side effectsusceptibility. Administration can be accomplished via single or divideddoses.

IV. Method of Treatment

In another aspect, the present invention provides a method of treatingan ALK- or FAK-mediated disorder or condition in a subject comprising:administering to the subject a compound of formula (I) or apharmaceutically acceptable salt form thereof. In another aspect, thepresent invention provides a compound of formula (I) or apharmaceutically acceptable salt form thereof for use in treating anALK- or FAK-mediated disorder or condition in a subject. In anotheraspect, the present invention provides a compound of formula (I) or apharmaceutically acceptable salt form thereof for use in the preparationof a medicament for treating an ALK- or FAK-mediated disorder orcondition in a subject. Preferably, the compound of formula (I) or apharmaceutically acceptable salt form thereof is administered to thesubject as a pharmaceutical composition comprising a pharmaceuticallyacceptable excipient. Preferably, the compound of formula (I) or apharmaceutically acceptable salt form thereof is administered to thesubject in a therapeutically effective amount. In one embodiment, theALK- or FAK-mediated condition or disorder is cancer. In anotherembodiment, the ALK- or FAK-mediated condition or disorder is selectedfrom anaplastic large cell lymphoma (ALCL), non-small cell lung cancer(NSCLC), neuroblastoma, glioblastoma, prostate cancer, squamous cellcarcinoma (SCC), and breast cancer. In certain embodiments, the ALK- orFAK-mediated condition or disorder is selected from ALK-positive ALCL,EML4-ALK-positive NSCLC, neuroblastoma, glioblastoma,androgen-independent prostate cancers, breast cancers, and head and necksquamous cell carcinomas (HNSCCs). In certain embodiments, the ALK- orFAK-mediated condition or disorder is selected from ALK-positive ALCL,EML4-ALK-positive NSCLC, neuroblastoma, androgen-independent prostatecancers, breast cancers, and HNSCCs. In certain embodiments, the ALK- orFAK-mediated condition or disorder is selected from ALK-positive ALCL,EML4-ALK-positive NSCLC, neuroblastoma, and glioblastoma. In certainembodiments, the ALK- or FAK-mediated condition or disorder is selectedfrom ALK-positive ALCL, EML4-ALK-positive NSCLC, and neuroblastoma. Incertain embodiments, the ALK- or FAK-mediated condition or disorder isselected from ALK-positive ALCL and EML4-ALK-positive NSCLC. In certainembodiments, the ALK- or FAK-mediated condition or disorder is selectedfrom androgen-independent prostate cancers, breast cancers, and HNSCCs.In certain embodiments, the ALK- or FAK-mediated condition or disorderis an ALK-mediated condition or disorder. In certain embodiments, theALK- or FAK-mediated condition or disorder is a FAK-mediated conditionor disorder. In certain embodiments, the ALK- or FAK-mediated conditionor disorder is a myofibroblastic tumor. In certain embodiments, the ALK-or FAK-mediated condition or disorder is a myofibroblastic tumor withTPM3-ALK or TPM4-ALK oncogenes. In certain embodiments, the ALK- orFAK-mediated condition or disorder is a myofibroblastic tumor withTPM3-ALK oncogenes. In certain embodiments, the ALK- or FAK-mediatedcondition or disorder is a myofibroblastic tumor with TPM4-ALKoncogenes.

The ALK- or FAK-mediated disorder or condition can be treatedprophylactically, acutely, or chronically using compounds of the presentinvention, depending on the nature of the disorder or condition.Preferably, the subject in each of these methods is human.

In another embodiment, the present invention provides a method oftreating a proliferative disorder in a subject, comprising administeringto the subject a compound of formula (I) or a pharmaceuticallyacceptable salt form thereof. In another aspect, the present inventionprovides a compound of formula (I) or a pharmaceutically acceptable saltform thereof for use in treating a proliferative disorder in a subject.In another aspect, the present invention provides a compound of formula(I) or a pharmaceutically acceptable salt form thereof for use in thepreparation of a medicament for treating a proliferative disorder in asubject. Preferably, the compound of formula (I) or a pharmaceuticallyacceptable salt form thereof is administered to the subject in apharmaceutical composition comprising a pharmaceutically acceptableexcipient. Preferably, the compound of formula (I) or a pharmaceuticallyacceptable salt form thereof is administered to the subject in apharmaceutically acceptable amount. In certain embodiments, theproliferative disorder is ALK- or FAK-mediated. In certain embodiments,the proliferative disorder is cancer. In certain embodiments, theproliferative disorder is selected from anaplastic large cell lymphoma(ALCL), non-small cell lung cancer (NSCLC), neuroblastoma, glioblastoma,prostate cancer, squamous cell carcinoma (SCC), and breast cancer. Incertain embodiments, the proliferative disorder is selected fromALK-positive ALCL, EML4-ALK-positive NSCLC, neuroblastoma, glioblastoma,androgen-independent prostate cancers, breast cancers, and head and necksquamous cell carcinomas (HNSCCs). In certain embodiments, theproliferative disorder is selected from ALK-positive ALCL,EML4-ALK-positive NSCLC, neuroblastoma, androgen-independent prostatecancers, breast cancers, and HNSCCs. In certain embodiments, theproliferative disorder is selected from ALK-positive ALCL,EML4-ALK-positive NSCLC, neuroblastoma, and glioblastoma. In certainembodiments, the proliferative disorder is selected from ALK-positiveALCL, EML4-ALK-positive NSCLC, and neuroblastoma. In certainembodiments, the proliferative disorder is selected from ALK-positiveALCL and EML4-ALK-positive NSCLC. In certain embodiments, theproliferative disorder is selected from androgen-independent prostatecancers, breast cancers, and HNSCCs.

The proliferative disorder can be treated prophylactically, acutely, orchronically using compounds of the present invention, depending on thenature of the disorder or condition. Preferably, the subject in each ofthese methods is human.

In therapeutic applications, the compounds of the present invention canbe prepared and administered in a wide variety of oral and parenteraldosage forms. Thus, the compounds of the present invention can beadministered by injection, that is, intravenously, intramuscularly,intracutaneously, subcutaneously, intraduodenally, or intraperitoneally.In certain embodiments, the compounds of the present invention areadministered intravenously or subcutaneously. Also, the compoundsdescribed herein can be administered by inhalation, for example,intranasally. Additionally, the compounds of the present invention canbe administered transdermally. In another embodiment, the compounds ofthe present invention are delivered orally. The compounds can also bedelivered rectally, bucally or by insufflation.

Determination of the proper dosage for a particular situation is withinthe skill of the practitioner. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstances is reached. For convenience, thetotal daily dosage may be divided and administered in portions duringthe day, if desired. A typical dose is about 1 mg to about 1,000 mg perday, such as about 5 mg to about 500 mg per day. In certain embodiments,the dose is about 10 mg to about 300 mg per day, such as about 25 mg toabout 250 mg per day.

V. Chemistry

CEP-28122

(1S,2S,3R,4R)-3-[5-chloro-2-((S)-1-methoxy-7-morpholin-4-yl-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino)-pyrimidin-4-ylamino]-bicyclo[2.2.1]hept-5-ene-2-carboxylicacid amide (CEP-28122) is prepared as described in Example 882 of WO2008/051547 (Ahmed et al.).

CEP-37440

The synthesis of2-(5-chloro-2-{(S)-6-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino}-pyrimidin-4-ylamino)-N-methyl-benzamidecan be carried out according FIG. 1, following the procedures outlinedin Steps 1-8.

Step 1: 5-Methoxy-1-methylene-1,2,3,4-tetrahydro-naphthalene

To a slurry of 5-Methoxy-3,4-dihydro-2H-naphthalen-1-one (25 g, 0.14mol) and methyltriphenylphos-phonium iodide (1.13 eq) in THF (250 mL) atRT was added potassium t-butoxide (1.6 eq) at such a rate as to maintaina temperature no higher than warm to the touch. The reaction was stirredfor one hour and concentrated. The reaction was then azeotroped withthree volumes of hexane to remove excess t-butanol. Fresh hexane wasadded the solution was let to stand overnight to effect trituration. Thered-brown solid was removed by filtration and the filtrate was washedtwice with water and was concentrated. Purification by chromatography onISCO (330 g SiO2 cartridge: stepwise hexane and then DCM) affords thetitle compound as a pale yellow oil (24 g, 99%). 1H-NMR (400 MHz, CDCl₃)7.29 (d, J=8.0 Hz, 1H), 7.15 (t, J=8.0 Hz, 1H), 6.76 (d, J=8.0 Hz, 1H),5.49 (s, 1H), 4.98 (s, 1H), 3.85 (s, 3H), 2.77 (t, J=6.4 Hz, 2H),2.53-2.50 (m, 2H), 1.93-1.87 (m, 2H).

Step 2: 1-Methoxy-5,7,8,9-tetrahydro-benzocyclohepten-6-one

5-Methoxy-1-methylene-1,2,3,4-tetrahydro-naphthalene (23.8 g, 0.137 mol)in 150 mL MeOH added in one portion to freshly prepared solution ofthallium(III)nitrate trihydrate (1.0 eq) in 300 mL MeOH. Stirred oneminute and 400 mL chloroform added. The solution was filtered and theorganics partitioned between dichloromethane and water. The organicswere dried (MgSO4) and concentrated. Purification by chromatography(ISCO, 330 g silica cartridge; stepwise elution hexane (5 min) then 7minute gradient to 100% dicloromethane (20 min) affords the titlecompound as the most polar of the products as a pale yellow oil (26 g,97%). 1H-NMR (400 MHz, CDCl₃) 7.16 (t, J=7.9 Hz, 1H), 7.84 (d, J=8.3 Hz,1H), 6.79 (d, J=7.5 Hz, 1H), 3.84 (s, 3H), 3.73 (s, 2H), 3.05-3.01 (m,2H), 2.55 (t, J=7.0 Hz, 2H), 2.01-1.96 (m, 2H). LC/MS (ESI+) m/z=191(M+H)+

Step 3: 1-Methoxy-2-nitro-5,7,8,9-tetrahydro-benzocyclohepten-6-one

To potassium nitrate in acetonitrile (50 mL) and trifluoroaceticanhydride (100 mL) at 0° C. was added dropwise1-methoxy-5,7,8,9-tetrahydro-benzocyclohepten-6-one (25 g, 0.131 mol) in50 mL acetonitrile. The reaction was stirred for 2.5 hours while warmingto RT. The reaction was concentrated without heat on a rotaryevaporator. MeOH was added and stirred briefly. Reconcentrated andworked-up by partitioning between dichloromethane and sat. sq. sodiumbicarbonate solution. The organic layer was separated and dried(Mg2SO4), concentrated and purified by chromatography ISCO (330 g silicacartridge: gradient elution −10 to 50% EA:HEX over 60 minutes) affordingtwo isomers. The title compound was the later eluting (10.7 grams, 34.6%yield). 1H-NMR (400 MHz, CDCl₃) 7.70 (d, J=8.3 Hz, 1H), 7.06 (d, J=8.3Hz, 1H), 3.92 (s, 3H), 3.80 (s, 2H), 3.13-3.09 (m, 2H), 2.60 (t, J=7.0Hz, 2H), 2.10-2.03 (m, 2H).

Step 4:2-[4-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)-piperazin-1-yl]-ethanol

1-Methoxy-2-nitro-5,7,8,9-tetrahydro-benzocyclohepten-6-one (15.09 g,64.15 mmol) in methylene chloride (870 ml) treated with2-Piperazin-1-yl-ethanol (3 eq) followed by acetic acid (10 eq). Themixture was stirred at 50° C. for 2 hrs and cooled to 0° C. and sodiumtriacetoxyborohydride (4 eq) was added, then warmed to RT and stirred.After a few hours starting material was still present. Added 0.4 eqfurther of sodium triacetoxyborohydride, then again after 6 hours.Stirred overnight. Poured into a solution of sat. aq. Sodium bicarbonateand ice and made basic to pH 10 with 1N sodium hydroxide, extracted 2×dichloromethane, dried MgSO4, filtered and concentrated. This materialwas taken up into ethanol and HO/ethanol was added. The resultingprecipitate was triturated for 2 hours then filtered. The solid wasfree-based using NaOH followed by sodium bicarbonate and extracted intodichloromethane to give the title compound (19 g, 85% yield). 1H-NMR(400 MHz, CDCl₃) 7.56 (d, J=8.2 Hz, 1H), 7.00 (d, J=8.2 Hz, 1H), 3.82(s, 3H), 3.63-3.06 (m, 2H), 3.29-3.24 (m, 1H), 3.00-2.86 (m, 3H),2.72-2.67 (m, 2H), 2.60-2.51 (m, 8H), 2.46-2.37 (m, 2H), 2.12-2.07 (m,2H), 1.87-1.78 (m, 1H), 1.37-1.29 (m, 1H). LC/MS (ESI+) m/z=350 (M+H)+

Step 5:2-[4-(2-Amino-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)-piperazin-1-yl]-ethanol

2-[4-(1-Methoxy-2-nitro-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)-piperazin-1-yl]-ethanol(19.0 g, 54.4 mmol) was split into two batches and dissolved in a totalof Ethanol (232 mL). 10% Pd/C (1.74 g, 1.64 mmol) was divided in halfand the reaction was hydrogenated for 3-4 hours at 50 psi. Each reactionmixture was filtered through celite to remove Pd. The filtrates werecombined and then concentrated and the title compound isolated as afoamy solid (17.25 g, 99% yield). 1H-NMR (400 MHz, CDCl₃) 6.76 (d, J=7.9Hz, 1H), 6.53 (d, J=7.9 Hz, 1H), 3.72 (broad s, 3H), 3.71 (s, 3H), 3.64(t, J=5.4 Hz, 2H), 3.26-3.20 (m, 1H), 2.84-2.72 (m, 5H), 2.62-2.56 (m,8H), 2.42-2.35 (m, 2H), 2.40-2.37 (m, 1H), 1.81-1.74 (m, 1H), 1.70(broad s, 1H), 1.41-1.33 (m, 1H). LC/MS (ESI+) m/z=320 (M+H)+

Step 6:2-[4-((S)-2-Amino-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)-piperazin-1-yl]-ethanol

34 grams of racemic2-[4-(2-Amino-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)-piperazin-1-yl]-ethanolwere separated using SFC (supercritical fluid CO₂) chromatography usinga Chiralcel OJ-H (3×15 cm) 808041 column with 15% methanol(0.2%DEA)/CO2, 100 bar eluent at 80 mL/min flow rate monitoring thewavelength of 220 nm with an injection volume: 0.5 mL, 20 mg/mL ethanol.16.9 grams of the (R)-enantiomer and 17 grams of the titled compoundwere isolated with a chemical purity >99% and an enantiomeric excess(ee) >99% (measured using a Chiralcel OJ-H analytical column). NMR andmass were equivalent to the racemic material. The absolute configurationof the first eluting isomer was unambiguously assigned as the(R)-configuration via small-molecule X-ray using anomalous dispersion ofthe bis-p-bromobenzyl derivative: 4-bromo-benzoic acid2-{4-[(R)-2-(4-bromo-benzoylamino)-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl]-piperazin-1-yl}-ethylester. Thus, the second eluting enantiomer was determined to be(S)-configuration.

Step 7: 2-(2,5-Dichloro-pyrimidin-4-ylamino)-N-methyl-benzamide

2-Amino-N-methyl-benzamide (24.4 g, 0.16 mol) in DMF (0.5 L) was added2,4,5-Trichloro-pyrimidine (39 g, 1.3 eq) and Potassium carbonate (1.3eq). Stirred under argon at 75° C. for 5 hrs and then at RT overnight.Poured into 1 L water and precipitate isolated by filtration and washed1:1 acetonitrile:water followed by drying in air stream and under vacuumto afford the title compound as a yellow solid (38 g, 78% yield). 11.70(s, 1H), 8.74 (d, J=8.2 Hz, 1H), 8.24 (s, 1H), 7.59 (d, J=8.4 Hz, 1H),7.53 (d, J=8.8 Hz, 1H), 7.16 (t, J=8.4 Hz, 1H), 6.28 (s, 1H), 3.06 (d,J=4.7 Hz, 3H).

Step 8:2-(5-Chloro-2-{(S)-6-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino}-pyrimidin-4-ylamino)-N-methyl-benzamide

To a sealed vessel2-[4-((S)-2-Amino-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-6-yl)-piperazin-1-yl]-ethano(2.69 g, 8.41 mmol) and2-(2,5-Dichloro-pyrimidin-4-ylamino)-N-methyl-benzamide (2.00 g, 6.73mmol) were combined in 1-methoxy-2-propanol (120 mL, 1200 mmol) followedby the addition of Methanesulfonic acid (2.44 mL, 37.7 mmol). Thereaction was then heated at 90° C. for 18 hours. The reaction mixturewas added to a separatory funnel and diluted with sat. bicarb until acloudy ppt formed. This was extracted with dichloromethane 3×. Theorganic layer was then washed with brine, dried over MgSO4, filtered andconcentrated. The residue was pumped dry then chromatographed on ISCOflash column. It was injected in dichloromethane onto a normal phasecolumn and eluted on a gradient of 0-10% (dichloromethane:10% NH4OH inMeOH). The desired product eluted around 9-10% and the 10% gradient washeld until product eluted completely. Mixed fractions concentrated andwere chromatographed on the Gilson reverse-phase HPLC gradient elution0-40% CH₃CN. Chromatogrpahy was repeated using normal phase silica andreverse phase HPLC to effect further purification as desired. Followingneutralization and concentration of all the material, the resultingsolid was obtained by taking the foam up into EtOAc and concentrating todryness several times to give the title compound (1.1 g, 28%). 11.02 (s,1H), 8.69 (d, J=8.9 Hz, 1H), 8.13 (s, 1H), 8.08 (d, J=8.4 Hz, 1H),7.59-7.50 (m, 2H), 7.41 (s, 1H), 7.13 (t, J=7.4 Hz, 1H), 6.91 (d, J=8.1Hz, 1H), 6.21 (s, 1H), 3.74 (m, 3H), 3.66-3.63 (m, 2H), 3.29-3.23 (m,1H), 3.06 (d, J=4.3 Hz, 3H), 2.92-2.72 (m, 5H), 2.66-2.55 (m, 8H),2.48-2.39 (m, 2H), 2.16-2.10 (m, 2H), 1.87-1.77 (m, 1H), 1.42-1.32 (m,1H). LC/MS (ESI+) m/z=580 (M+H)+

CEP-37440 Amorphous HCl Salt

2-(5-Chloro-2-{6-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino}-pyrimidin-4-ylamino)-N-methyl-benzamidehydrochloride:2-(5-Chloro-2-{6-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino}-pyrimidin-4-ylamino)-N-methyl-benzamide(4.90 g, 8.45 mmol) and 2.5 M of HCl in ethanol (13.5 mL, 33.8 mmol)were heated until they dissolved in ethanol (164 mL). The reaction wasconcentrated two times from ethanol, then warmed in a small amount ofethanol until completely dissolved. This solution was allowed to coolslowly with a stirring (<100 rpm). A solid preciptate formed quicklybefore the solution had cooled. This mixture was allowed to stir untilambient temperature was achieved and then filtered. The solid was washedwith ethanol followed by ether then directly pumped dry under high vacto give2-(5-chloro-2-{6-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-1-methoxy-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-ylamino}-pyrimidin-4-ylamino)-N-methyl-benzamidehydrochloride (5.3 grams, quantitative yield). 1H-NMR (MeOD, 400 MHz) δ8.55 (s, 1H), 8.17 (s, 1H), 7.80 (d, J=7.7 Hz, 1H), 7.55 (t, J=6.8 Hz,1H), 7.46 (broad s, 1H), 7.36 (t, J=7.2 Hz, 1H), 7.23 (d, J=8.5 Hz, 1H),4.00-3.95 (m, 4H), 3.83-3.72 (m, 5H), 3.73 (s, 3H), 3.65-3.59 (m, 2H),3.47-3.38 (m, 5H), 2.95 (s, 3H), 2.72-2.65 (m, 1H), 2.44-2.38 (m, 1H),2.29-2.28 (m, 1H), 2.19-2.12 (m, 1H), 1.59-1.49 (m, 1H). LC/MS (ESI+)m/z=580 (M+H)+.

CEP-37440 Salt Screening

Salt screening experiments were performed (a) in methanol using 27different acids (acetic acid, ascorbic acid, benzenesulfonic acid,citric acid, D—glucuronic acid, DL glutamic acid, DL—lactic acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, fumaric acid, glycolicacid, hippuric acid, hydrobromic acid (48% aq), hydrochloric acid (2.5M,EtOH), L—aspartic acid, L—tartaric acid, L—pyroglutamic acid,lactobionic acid, maleic acid, malic acid, malonic acid,naphthalene-2-sulfonic acid, ortho phosphoric acid, p-toluenesulfonicacid monohydrate, propionic acid, succinic acid, and sulfuric acid), (b)in dichloromethane and tetrahydrofuran using 9 different acids(benzenesulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid,hydrobromic acid (48% aq), naphthalene-2-sulfonic acid, o-phosphoricacid (85%), sulfuric acid, p-toluenesulfonic acid, and hydrochloric acid(ethanol)), and (c) in chloroform, acetone, ethyl acetate, and1-propanol using benzenesulfonic acid, hydrobromic acid (48% aq),o-phosphoric acid, sulfuric acid, p-toluenesulfonic acid, andhydrochloric acid (ethanol)). Although many of the experiments providedsalt forms, only a few of the experiments resulted in crystalline salts,and only two salt forms were crystalline, stable, and reproducible: atri-benzenesulfonate form and a trihydrochloride dihydrate form. From apharmaceutical perspective the trihydrochloride dihydrate is favoredover the tri-benzenesulfonate because HCl is a Class 1 acid additionsalt that is generally recognized as safe (GRAS), whereasbenzenesulfonic acid is a Class 2 acid addition salt that is not GRAS(see Stahl, H. P. & Wermuth, C. G., Editors, 2002 Handbook ofPharmaceutical salts; Properties, Selection, and Use. VwerlagHelveticaChimica Acta and Wiley-VCH). In addition to being less toxic,HCl has a lower molecular weight than benezensulfonic acid, whichprovides a higher API/acid ratio and a lower effective dose.

Powder X-ray diffraction patterns were recorded on a PANalytical X PertPro diffractometer equipped with an X'celerator detector using CuK_(α)radiation at 45 kV and 40 mA. K_(α1) radiation was obtained with ahighly oriented crystal (Ge111) incident beam monochromator. A 10 mmbeam mask, and fixed (¼°) divergence and anti-scatter (⅛°) slits wereinserted on the incident beam side. A fixed 5 mm receiving slit and a0.04 radian Soller block were inserted on the diffracted beam side. TheX-ray powder pattern scan was collected from ca. 2 to 40° 2θ with a0.0080° step size and 96.06 sec counting time which resulted in a scanrate of approximately 0.5°/min. The sample was spread on silicon zerobackground (ZBG) plate for the measurement. The sample was rotated usinga PANalytical PW3064 Spinner (15 revolutions/min.). Measurement of theSi reference standard before the data collection resulted in values for20 and intensity that were well within the tolerances of 28.44<2θ<28.50and significantly greater than the minimum peak height of 150 cps.

CEP-37440 Tribenzenesulfonate

CEP-37440 free base (500 mg, 0.862 mmoles) was dissolved in 6 mL oftetrahydrofuran by stirring at room temperature for 5 minutes. Thissolution was added one mL at a time to benzenesulfonic acid (545.6 mg,3.45 mmoles) in a glass 20 mL scintillation vial (16×60 mm). The samplewas mixed using a stirring bar during the addition (at roomtemperature). An oil plus a solid was noted when all of the CEP-37440solution was added. The sample was stirred at 5-7° C. for 19 hours on aHEL Polyblock™ unit. The solid was isolated by suction filtration. Thesolid was dried at 50° C. under house vacuum for 5 hours to give 835 mg(80% recovery) of slightly yellow solid. The HPLC purity, assay, andcompound color were improved by suspending 616 mg in 1.5 mL of water andstirring the resulting slurry for 30 minutes at room temperature. Thesolid was isolated by suction filtration and the solid on the filter padwas washed with 1 mL of water. The white solid was dried at 50° C. underhouse vacuum for 68 hours to yield 406 mg (67% recovery). Thetribenzenesulfonate salt has a water solubility of about 20-33 mg/mL.

The X-ray diffraction data characteristic of the crystallinetribenzenesulfonate salt is shown in Table 1 and FIG. 2.

TABLE 1 Higher Relative Intensities with Two Theta Positions andd-Spacings for the XRPD Pattern of the CEP-37440 tribenzenesulfonatesalt No. Pos. [2θ.] d-spacing [Å] Rel. Int. [%] 1 6.85 12.897 8 2 7.6211.593 100 3 8.01 11.031 9 4 10.77 8.209 6 5 12.07 7.324 5 6 13.11 6.74720 7 13.34 6.630 5 8 13.76 6.430 44 9 14.05 6.296 21 10 14.60 6.061 9 1115.25 5.805 6 12 15.47 5.724 5 13 15.57 5.686 8 14 15.74 5.625 6 1516.02 5.528 2 16 16.66 5.317 3 17 17.10 5.181 40 18 17.59 5.038 4 1917.86 4.962 24 20 18.10 4.898 47 The highest peak (intensity 100%) isset in bold letters.

CEP-37440 Trihydrochloride Dihydrate

In a 20 mL scintillation vial containing 200 mg of CEP-37440 free basewas added n-butyl alcohol (5 mL) at room temperature. After stirring for5 minutes, the free base was in solution. HCl (2.5 M in EtOH, 0.435 mL,3.15 eq) was then added resulting in the immediate precipitation ofwhite solids (amorphous HCl salt). The resulting slurry was heated to85° C. over 20 minutes. (Note: at approximately 60° C., no solidsremained out of solution.) Stirring the solution between 80 and 85° C.resulted in self nucleation after 30 minutes which resulted in furtherprecipitation of white solids. After stirring the reaction for a totalof 2 hours, the resulting slurry was cooled to 5° C. over 1 hour. The0-5° C. slurry was stirred for an additional hour then filtered, washingwith minimal cold n-butyl alcohol. The solids were then dried at 55° C.overnight. Upon standing to RH 30-70% air, 208 mg of CEP-37440-3HCl-2H₂Owere obtained.

The X-ray diffraction pattern characteristic of the crystallinetrihydrochloride dihydrate salt is shown in Table 2 and FIG. 3. The saltis stable at 30-80% relative humitidy, but reversibly converts to atrihydrochloride monohydrate form below 30% RH and irreversibly convertsto a highly hydrated amorphous form above 80% RH.

TABLE 2 Higher Relative Intensities with Two Theta Positions andd-Spacings for the XRPD Pattern of the CEP-37440 trihydrochloridedihydrate salt No. Pos. [2θ.] d-spacing [Å] Rel. Int. [%] 1 5.42 16.306100 2 5.91 14.940 39 3 7.02 12.575 8 4 8.86 9.967 77 5 10.80 8.184 46 611.06 7.995 14 7 11.79 7.498 33 8 14.06 6.292 64 9 14.22 6.223 26 1014.72 6.014 45 11 15.70 5.641 15 12 17.02 5.204 44 13 17.52 5.059 83 1417.76 4.990 14 15 18.51 4.789 56 16 19.13 4.635 3 17 20.56 4.316 20 1820.79 4.269 17 19 21.12 4.204 24 20 21.66 4.099 17 The highest peak(intensity 100%) is set in bold letters.

VI. Biology

13-Week Oral Toxicity and Toxicokinetic Study of CEP-28122 inSprague-Dawly Rats, Including a 4-Week Recovery Period

Three treatment groups of 20 rats/sex were administered CEP-28122 atrespective dose levels of 30, 75, and 150 mg free base/kg/day(administered as the mono-methanesulfonic acid, mono-HCl salt form). Oneadditional group of 20 animals/sex served as the control and receivedthe vehicle, distilled water. The drug or vehicle was administered toall groups via oral gavage, once a day for 91 consecutive days, at adose volume of 10 mL/kg/dose. Following the dosing period, fiveanimals/sex/group were maintained for a 4-week recovery period.Additionally, one group of three animals/sex and three groups of nineanimals/sex/group served as toxicokinetic (TK) animals and received thevehicle or drug in the same manner and at the same dose levels as themain study groups.

Observations for morbidity, mortality, injury, and the availability offood and water were conducted at least twice daily for all animals.Clinical observations were conducted on main study animals weekly. Bodyweights were measured and recorded for all animals prior to initiationof dosing (week −1) and weekly during the study. Food consumption wasmeasured and recorded for main study animals weekly. Ophthalmoscopicexaminations were conducted on main study animals pretest and prior tothe terminal and recovery necropsies. Blood samples for designatedclinical pathology evaluations were collected from main study animals atthe end of week 4 and at the terminal and recovery necropsies. Urinesamples for urinalysis evaluations were collected from main studyanimals prior to the terminal and recovery necropsies. Blood samples fordetermination of the plasma concentrations of the drug were collectedfrom designated TK animals at designated time points on day 1, week 4,and week 13. After the final blood collection, the TK animals wereeuthanized and the carcasses were discarded. At the end of the terminaland recovery periods, necropsy examinations were performed, organweights were recorded, and selected tissues were microscopicallyexamined.

13-Week Oral Toxicity and Toxicokinetic Study of CEP-28122 in CynomolgusMonkeys with a 6-Week Recovery Period

CEP-28122 solutions were prepared weekly by dissolution of a CEP-28122mono-methanesulfonic acid, mono-HCl salt in sterile water for injection(SWFI) to achieve the desired dose concentration levels.

Forty experimentally naive cynomolgus monkeys (20 males and 20 females),2.5 to 4.4 years of age for the males and 2.5 to 4.0 years of age forthe females, and weighing 2.1 to 3.3 kg for the males and 2.0 to 3.1 kgfor the females at the outset (Day −1) of the study, were assigned toone of three dose groups and a vehicle control group. Five animals/sexwere assigned to each dose group and were given daily oral doses of 0(vehicle), 20, 40 or 80 mg/kg for up to 91 days. Due to the occurrenceof seizures in several animals given 80 mg/kg/day during the first twoweeks of dosing, the high dose was lowered to 60 mg/kg/day. Threeanimals/sex/group were scheduled for termination at the end of thedosing period (Day 92) and two animals/sex/group were assigned to a6-week non-dosing recovery phase and were terminated on Day 134.

The animals were evaluated for changes in clinical signs (cage sideobservations and food consumption [twice daily], and post doseobservations [1-2 hours post dose on each dosing day]), body weight(Weeks −2 and −1, and weekly thereafter starting on Day 7, and prior tonecropsy), electrocardiograms, ophthalmic examinations, and bloodpressure measurements (prestudy and in Weeks 1, 4, and 13),echocardiographic measurements (Week 11), clinical pathology indices,including serum chemistry, hematology, and coagulation (within one weekprior to the initiation of dosing and near the end of Weeks 1, 4, 10,and 13; and from remaining animals near the end of the recovery period;and troponin I (prestudy; prior to dose and 2, 4, and 24 hours followinga dose in Weeks 1, 4, and 13, and at a single time point near the end ofthe recovery phase). Urine samples for urinalysis only were obtained bybladder puncture during necropsy. Urine samples for urinalysis and urinechemistry analysis were also obtained by drainage from specialstainless-steel cage pans in Weeks—1, 1, 4, and 13, and from theremaining animals near the end of the recovery period.

Blood samples were collected for toxicokinetic analysis at various timepoints on Day 1 and during Weeks 4 and 13. Twenty-one animals(3/sex/Group 1, 2 males/3 females/Group 2, 3 males/2 females/Group 3 and2 males/3 females/Group 4) were euthanized one day after the last dose.The remaining 14 animals (2/sex each from Groups 1 and 2, 1 male/2females/Group 3, and 2 males/1 female/Group 4) were continued on studywithout further dosing, and euthanized approximately 6 weeks after thelast dose. At termination, a full necropsy was conducted on all animals,and tissues were collected, preserved, processed, and examinedmicroscopically by a Veterinary Pathologist certified by the AmericanCollege of Veterinary Pathologists.

4-Week Oral Toxicity and Toxicokinetic Study of CEP-28122 in CynomolgusMonkeys with a 4-Week Recovery Period

CEP-28122 (mono-methanesulfonate, mono-HCl salt) was administered viaoral gavage to groups of cynomolgus monkeys (5/sex/group) at dose levelsof 0 (vehicle control), 3, 10, 20 or 40 mg/kg/day. Following the 4-weektreatment period, 3 animals/sex/group were terminated and 2animals/sex/group entered a 4-week treatment-free recovery period.

The following parameters and end points were evaluated in this study:clinical signs (mortality/moribundity checks, cage side observations andfood consumption, and postdose observations), body weights, veterinaryphysical examinations, lung evaluations, ophthalmology,electrocardiography, blood pressure and heart rate measurements,clinical pathology parameters (hematology, coagulation, clinicalchemistry, urinalysis, urine chemistry, and troponin I analysis),toxicokinetic parameters, gross necropsy findings, organ weights, andhistopathologic examinations.

4-Week Oral Toxicity and Toxicokinetic Study of CEP-37440 inSprague-Dawley Rats with a 4-Week Recovery Period

Male and female rats (15/sex/group) were assigned to 4-treated groups,and a vehicle control group (pH-adjusted reverse osmosis water), and theCEP-37440 was administered as the trihydrochloride dihydrate salt asindicated in the following table. Animals were dosed via oral gavage.

No. of Animals Dose Level^(c,d) Group^(a) Subgroup^(b) Male Female(mg/kg/day) 1 (Control) 1 (Toxicity) 15 15 0 2 (Toxicokinetic) 3 3 0 2(Low) 1 (Toxicity) 15 15 1 2 (Toxicokinetic) 9 9 1 3 (Mid) 1 (Toxicity)15 15 3 2 (Toxicokinetic) 9 9 3 4 (Mid-High) 1 (Toxicity) 15 15 10 2(Toxicokinetic) 9 9 10 5 (High) 1 (Toxicity) 15 15 30 2 (Toxicokinetic)9 9 30 ^(a)Group 1 received vehicle control (pH-adjusted RO water) only.^(b)Toxicity animals designated for recovery sacrifice (up to fiveanimals/sex in Groups 1, 2, 3, 4, and 5) underwent 4 weeks of recoveryfollowing administration of the last dose. ^(c)Doses expressed as freebase. ^(d)The dose volume was 10 mL/kg.

Assessment of toxicity was based on mortality, clinical observations,body weights, body weight changes, food consumption, ophthalmicexaminations, and clinical and anatomic pathology. Blood samples werecollected for toxicokinetic evaluations.

4-Week Oral Toxicity and Toxicokinetic Study of CEP-37440 in CynomolgusMonkeys with a 4-Week Recovery Period

Male and female cynomolgus monkeys (Macaca fascicularis) were assignedto 4 groups (3-treated and a vehicle control group) consisting of 5monkeys/sex/group. Dose levels evaluated in this study were 0(pH-adjusted reverse osmosis water), 2.5, 7.5, and 20 mg/kg/day. Thedosing volume was 5 mL/kg. CEP-37440 was administered as thetrihydrochloride dihydrate salt form. After completion of the dosingphase, 3 monkeys/sex/group were euthanized and 2 monkeys/sex/groupcontinued on a treatment-free recovery phase for an additional 4 weeks.

Anti-Tumor Efficacy Studies—General Protocol

Tumor-bearing mice were randomized into different treatment groups (8-10mice/group) and were administered orally with either vehicle (PEG-400)or test compound formulated in PEG-400 at indicated doses (expressed asmg/kg equivalents of free base) and with indicated dosing frequency (bidor qid), with 100 μL per dosing volume. The length (L) and width (W) ofeach tumor was measured with a vernier caliper and the mouse body weightwas determined every 2-3 days. The tumor volumes were then calculatedwith the formula of 0.5236*L*W*(L+W)/2. Statistical analyses of tumorvolumes and mouse body weight were carried out using the Mann-WhitneyRank Sum Test. Plasma and tumor samples were obtained at 2 hours postfinal dose at each dose level, and the compound levels in plasma andtumor lysates were measured by LCMS/MS.

Anti-Tumor Efficacy in NPM-ALK Positive Sup-M2 and Karpas-299 ALCL TumorXenograft Models in Mice

CEP-37440 is administered po in PEG400 qd or bid to mice containingNPM-ALK positive Sup-M2 ALCL tumor xenografts for 12 days. Theanti-tumor efficacy of CEP-37440 (amorphous HCl salt) in a second, moreresistant NPM-ALK-positive ALCL (Karpas-299) tumor xenograft model inScid mouse was also evaluated.

Anti-Tumor Activity in EML4-ALK Positive (NCI-H2228 and NCI-H3122) NSCLCTumor Xenografts in Mice with Oral Dosing

Human lung cancer cell lines, NCI-H2228 and NCI-H1650 (ATCC, Manassas,Va.), and NCI-H3122 (kindly provided by Dr. Giorgio Inghirami, Univ. ofTorino, Italy), were grown in RPMI-1640 medium supplemented with 10%fetal bovine serum (FBS, Cat# SH3007003, Hyclone, Logan, Utah).NCI-H2228 cells harbor EML4-ALK variant 3a/b and NCI-H3122 cells containEML4-ALK variant 1, determined by fluorescence in situ hybridization andreverse-transcription-PCR as previously reported (Koivunen J P, MermelC, Zejnullahu K, Murphy C, Lifshits E, Holmes A J, et al. EML4-ALKfusion gene and efficacy of an ALK kinase inhibitor in lung cancer. ClinCancer Res 2008, 14:4275-8).

Generation of EML4-ALK-positive and EML4-ALK-negative NSCLC SubcutaneousTumor Xenografts in Scid Mice Female Scid/Beige mice (6-8 weeks,Taconic, Hudson, N.Y.) were maintained 5/cage in microisolator units ona standard laboratory diet (Teklad Labchow, Harlan Teklad, Madison,Wis.). Animals were housed under humidity- and temperature controlledconditions and the light/dark cycle was set at 12-hour intervals. Micewere quarantined at least 1 week prior to experimental manipulation. Allanimal studies were conducted under protocol (#03-023) approved by theInstitutional Animal Care and Use Committee (IACUC) of Cephalon, Inc.Briefly, EML4-ALK-positive and -negative NSCLC cells were collected andresuspended in RPMI-1640 medium at density of 5×107/mL. An aliquot (100μL) of the cell suspension (5×106 cells) was inoculated subcutaneouslyto the left flank of each mouse with a 23 g needle (23G1, Cat#305145,Becton Dickinson, Franklin, N.J.). The mice were monitored until thetumor xenograft volumes reached 200-500 mm3.

The tumor bearing mice were randomized into different treatment groups(8-10 mice/group) and were administered either vehicle (PEG-400) orCEP-37440 amorphous HCl salt formulated in PEG-400 at indicated doses,bid, with 100 μL per dosing volume. The length (L) and width (W) of eachtumor were measured with vernier calipers and the mouse body weight wasdetermined every two to three days. Tumor volumes were calculated withthe formula of 0.5236*L*W*(L+W)/2. Percent tumor growth inhibition (%TGI) was calculated as follows: (tumor volume of the control group atend of treatment—tumor volume of the treated group at the end oftreatment)/tumor volume of the control group at the end of treatment.Partial tumor regression (PR) was defined as the tumor volume of thetreated group at the end of treatment being less than that of thetreated group at the start of treatment. Complete tumor regression (CR)was defined as the tumor volume of the treated group at the end oftreatment being less than 5% of the tumor volume of the treated group asthe start of treatment. Statistical analyses of tumor volumes and mousebody weights were carried out with the Mann-Whitney Rank Sum Test.Plasma and tumor samples were obtained at 2 hours post final dose ateach dose level, and the compound levels in plasma and tumor lysateswere measured by LC-MS/MS

Anti-Tumor Efficacy Studies in Human Tumor Xenografts ofHormone-Independent Prostate Carcinoma, NSCL Carcinoma and HNSCCarcinoma

The human prostate carcinoma cell lines, CWR22 and PC3, and human headand neck squamous cell carcinoma cell line Detroit 562 were obtainedfrom American Tissue Culture Collection (ATCC, Manassas, Va.). CWR22cells were cultured in RPMI (ATCC, Cat #30-2001,) supplemented with 10%fetal bovine serum (FBS, Cat# SH3007003, Hyclone Laboratory Inc, Logan,Utah), PC3 were cultured in F12 medium (ATCC, Cat#30-2004,) supplementedwith 10% FBS, and Detroit 562 were cultured in EMEM (ATCC, Cat#30-2003,)supplemented with 10% FBS. Human non-small cell lung cancer cell lineHCC-827 and human breast cancer cell line BT474 were also purchased fromthe ATCC(Manassas, Va.) and cultured in RPMI (Cat#10-040, Mediatech Inc,Manassas, Va.) with 10% FBS. The rabbit phospho-FAK(Tyr397) (Cat#3283)and FAK antibodies (Cat#3285) were purchased from Cell SignalingTechnology (Beverly, Mass.).

Generation of Subcutaneous Human Tumor Xenografts in SCID/Beige or Nu/NuMice Female SCID/Beige (6-8 weeks, Taconic, Hudson, N.Y.) or Nu/Nu mice(6-8 weeks, Charles River Laboratory, Wilmington, Mass.) were maintained5/cage in microisolator units on a standard laboratory diet (TekladLabchow, Harlan Teklad, Madison, Wis.). Animals were housed underhumidity- and temperature-controlled conditions and the light/dark cyclewas set at 12-hour intervals. Mice were quarantined at least 1 weekprior to experimental manipulation. Experiments were approved (Protocol03-023) by the Institutional Animal Care and Use Committee of Cephalon,Inc. Briefly, the cells were collected and resuspended in RPMI medium atdensity of 5×107/mL and an aliquot (100 μL) of the cell suspension(4×106 or 5×106 cells) was inoculated subcutaneously to the left flankof each mouse with a 23 g needle (23G1, Cat#305145, Becton Dickinson,Franklin, N.J.). The mice were then monitored daily.

The tumor-bearing mice were randomized into different treatment groups(8-10 mice/group) and were administered orally with either vehicle(PEG-400) or CEP-37440 formulated in PEG-400 at indicated doses(expressed as mg/kg equivalents of free base) and with indicated dosingfrequency, with 100 μL per dosing volume. The length (L) and width (W)of each tumor was measured with a vernier caliper and the mouse bodyweight was determined every 2-3 days. The tumor volumes were thencalculated with the formula of 0.5236*L*W*(L+W)/2. Statistical analysesof tumor volumes and mouse body weight were carried out using theMann-Whitney Rank Sum Test. Plasma and tumor samples were obtained at 2hours post final dose at each dose level, and the compound levels inplasma and tumor lysates were measured by LC-MS/MS. The TGI values werecalculated at the end of study by comparing the tumor volumes (TV) ofeach CEP-37440-treatment group with those of vehicle-treated group withthe following formula: [1−(the last day TV of compound-treated group/thelast day TV of vehicle treated group)]*100.

VII. Results

Biological data for CEP-37440 and CEP-28122 are summarized in Table 3and presented and discussed below.

TABLE 3 Summary of data for CEP-37440 and CEP-28122 Activity CEP-37440CEP-28122 ALK enzymatic IC₅₀ (nM) 3.5 3 ALK cellular IC₅₀ (nM) 40 30 ALKcellular IC₅₀ in 75% murine 200 300 plasma (nM) ALK cellular IC₅₀ in 75%human 120 300 plasma (nM) FAK enzymatic IC₅₀ (nM) 2.3 29.5 FAK cellularIC₅₀ (nM) 82 944 Enzymatic IR IC₅₀ (nM) 66 996 Enzymatic IR/ALK IC₅₀ratio 19 332 Cellular IR IC₅₀ (nM) 2000 >10,000 Cellular IR/ALK IC₅₀ratio 50 >333 Kinase selectivity - 442 kinases (Ambit S(80): 0.131;S(90): S(80): 0.172; S(90): KINOMEscan profiling @ 1 μM) 0.084; S(99):0.016 0.125; S(99): 0.016 Receptor selectivity (Cerep) (Ki, μM)Histamine H2 = 5.2; Adrenergic α_(1B) (R), 2.2; Muscarinic: M1 = 2.46,Adrenergic α_(1D) (H), 1.1 M2 = 2.0, M3 = 3.5, M4 = 0.46; Neurokinin:NK1 = 1.2; Serotonin: 5-HT1B = 3.3; DA transporter = 2.1 hERG patchclamp (IC₅₀, μM) >10 >10 Equilibrium solubility at pH 2/pH7.4 >1/0.28 >3/0.013 (mg/mL) cLog D_(7.4) 2.95 3.9 In vitro metabolicstability (t_(1/2), min) >40 (M), >40 (R), 21 13.2 (M), 6.7 (R), 19.9(Mo), >40 (H) (D), <5 (Mo), 10.3 (H) CYP inhibition (IC₅₀, μM) 1A2: >30;2C9: >30; 1A2: >30; 2C9: 1.3; 2C19: >20; 2C19: 6.8; 2D6: >30; 3A4: 52D6: 17.3; 3A4: 1.1 CYP3A4 induction in vitro (fold) 1.5 @ 10 μM 0.95 @0.3 μM; 1.3 @ 3.0 μM; 2.0 @ 10 μM Caco-2 P_(app) × 10⁻⁸ cm/s (PDR) 1.26(PDR: 4.7) 12.6 (PDR: 3.6) Protein binding (% bound) 85 (M), 94 (D), 95(H) 95 (M), 96 (D), 99.2 (H) Rat bioavailability >42% 25% Mousebioavailability 102% (CD-1), 99% (Scid) 51% (CD-1), 71% (Scid) Monkeybioavailability 50%@10 mg/kg, 16%@10 mg/kg, 74%@30 mg/kg 37%@30 mg/kgmetabolic stability/protein binding: (M) mouse, (R) rat, (Mo) monkey,(H) human, (D) dog mouse and monkey bioavailability experiments forCEP-37440 were performed with the CEP-37440 amorphous HCl salt

These data indicate that both CEP-37440 and CEP-28122 are potent ALKinhibitors. However, CEP-37440 is advantageous in comparison toCEP-28122 in that CEP-37440 is a far more potent FAK inhibitor, hasreduced protein binding and increased activity in human plasma,increased intrinsic solubility (facilitates absorption), reducedlipophilicity (higher lipophilicity has been associated with increasedtoxicity), increased metabolic stability (facilitates higher bloodconcentration at lower doses, which reduces xenobiotic burden on thebody—less exposure to drug and metabolites and less pressure on themetabolic system, e.g., liver), has reduced capacity for drug-druginteraction due to diminished P450 inhibition, particularly with respectto CYP3A4 and CYP2C9 (reduced interference with normal metabolism andclearance of co-administered drugs), and possesses improved oralbioavailability and a lower clearance rate in vivo (higher bloodconcentrations at lower doses). The favorable properties of CEP-37440 incomparison to CEP-28122 are surprising and unexpected.

13-Week Oral Toxicity and Toxicokinetic Study of CEP-28122 inSprague-Dawley Rats, Including a 4-Week Recovery Period

There were no drug-related deaths during the study and no drug-relatedeffects on body weight, food consumption, or cardiac troponinconcentrations. Drug-related effects on hematology parameters werelimited to minimal, non-adverse reductions in erythrocytes, hemoglobinand hematocrit in females and minimal, non-adverse increases inplatelets in both sexes. Other statistical differences were observed andconsidered not meaningful due to the magnitude or direction change,and/or the lack of dose-dependency.

Platelets remained mildly elevated in males and females at 150 mg/kg/dayat recovery. Possible drug-related effects on coagulation parameterswere limited to prolongation of prothrombin time (PT) and partialthromboplastin time (APTT) in males that received 150 mg/kg/day andshortening of AP and APTT in females at all dose levels (values forfemales remained within expected ranges). There were no meaningfulchanges evident at the end of the recovery period.

Drug-related changes in clinical chemistry parameters were limited tomoderate, but inconsistent increases in alanine aminotransferase (ALT),total bilirubin, aspartate aminotransferase (AST), γ-glutamyltransferase(GGT), and sorbitol dehydrogenase (SDH) among males that received ≧75mg/kg/day. These effects were more pronounced at the end of the 13 weekdosing period than they were at the time of the 4-week interim samplecollection and were mostly attributable to effects in a single animal.AST and ALT also were increased in the females that received 150mg/kg/day but these effects were generally much lower magnitude thanobserved in the males. Notably, females also exhibited a statisticallysignificant, dose-dependently, and progressive increase in cholesterolat all dose levels. At the end of the recovery period, AST, ALT, and SDHremained elevated in individual males that had received 75 mg/kg/dayduring the dosing period. AST, ALT, and SDH remained moderatelyincreased in males that had received 150 mg/kg/day during dosing and, toa lesser magnitude, in the females that had received 150 mg/kg/dayduring dosing.

Total protein and globulins were dose-dependently increased in malesthat received ≧75 mg/kg/day and in females that received ≧30 mg/kg/day.Albumin also was statistically increased in females that received ≧75mg/kg/day, but values remained within expected ranges. Statisticalincreases in calcium were considered secondary to the increased albumin,and total protein. Other sporadic statistical differences were observedat termination and considered not meaningful due to the magnitude ordirection of change, and/or the lack of dose-dependency. There were noeffects of dosing on total protein, globulin, albumin, or calcium at theend of the recovery period.

Urine volumes increased and specific gravity decreased in males andfemales at 150 mg/kg/day at termination. No other remarkable changeswere observed in urinalysis parameters at termination or recovery.

Increased liver weights (relative to controls) were noted in femalesthat received ≧30 mg/kg/day and in males that received ≧75 mg/kg/day(Males also had decreased thymus weights relative to control at 75mg/kg/day). Increased kidney, spleen, and adrenal weights were noted inboth sexes at dosages ≧75 mg/kg/day. Heart weights also were modestlyincreased at dosages ≧75 mg/kg/day. However, because there were nocorrelative microscopic findings in the heart, the toxicologicalsignificance of this finding is not clear. Effects in the kidney, heartand spleen of males persisted to the end of the four-week recoveryperiod. There were no persistent drug-related effects evident infemales. At the end of the 13-week dosing period, brown or blackdiscoloration of the kidneys was observed macroscopically in males thatreceived 150 mg/kg/day and in females that received 75 mg/kg/day. Onemale that received 150 mg/kg/day had focus/foci on the kidneys whichcorrelated microscopically to tubular pigment at the end of recovery. Atthe end of the 13-week dosing period, drug-related microscopicalterations were evident in the kidneys, adrenal glands, liver, lung,brown adipose tissue, mesenteric lymph node, spleen, and thyroid glandsof animals that received ≧30 mg/kg/day. Findings in the adrenal glands,liver, lung, brown adipose tissue, mesenteric lymph node, spleen, andthyroid glands are not considered to be adverse due to low severity andsubstantial evidence of recovery during the 4-week recovery period.Progressive neuropathy, evident in the kidneys of animals that received≧75 mg/kg/day was considered to be adverse due to due to high prevalenceand persistence throughout the 4 week recovery period. Pigmentation,presumed to be accumulated drug, also was evident in several tissues atrecovery but is not considered to be adverse.

Minimal to mild swelling of lenticular fibers, especially in thesubcapsular axial cortex, was evident in recovery animals that received≧75 mg/kg/day during the dosing period. This finding corresponded withclinical cataract in these animals. Swelling of lens fibers; however,can be an artifact of fixation with Davidson's fluid resulting from cellswelling associated with acetic acid. Swelling of lens fibers in thesubcapsular cortical region was noted in treated animals (including theanimals listed above and other treated animals with no record ofclinical cataract) and, to a lesser extent, in some control animals inthis study. Additionally, swelling of lens fibers in a circumferential,anterior cortical pattern was noted in a few treated animals at 75 and150 mg/kg/day, but with no corresponding ophthalmoscopic findings, thisfinding was regarded as artifact secondary to Davidson's fixation. Forthese reasons, the swelling of lens fibers observed in this study wasinterpreted as artifact, and was not recorded as a microscopic finding.However, the possibility of a real antemortem change that was masked bythe artifacts cannot be completely excluded.

Based on the results noted above, the No Observable Adverse Effect Level(NOAEL) for this study was considered to be 30 mg/kg/day, limited byadverse histopathological effects in the kidney that persistedthroughout the 4-week recovery period at dosages ≧75 mg/kg/day.

13-Week Oral Toxicity and Toxicokinetic Study of CEP-28122 in CynomolgusMonkeys with a 6-Week Recovery Period

The administration of CEP-28122 via daily nasogastric gavage for 91consecutive days at dose levels of 20, 40, and 80/60 mg/kg resulted inseveral adverse drug-related events at all dose levels includingmorbidity and mortality in two animals dosed with 80/60 mg/kg and likelytwo additional animals (one dosed with 40 mg/kg and the other dosed with20 mg/kg). There was an additional animal that underwent early necropsy;however, this was not related to CEP-28122 administration. Theadministration of 80 mg/kg resulted in seizures for 2 animals on Days 9and/or 10 and led to a dose level reduction to 60 mg/kg in all animalswithin this group. There were no CEP-28122-related changes in foodconsumption, body weight, ophthalmic examinations, coagulationparameters, urinalysis parameters, and heart rate, and there were nodefinitive CEP-28122-related changes in blood pressure or troponin I.

The clinical history for animals that underwent early necropsy couldgenerally be divided into two categories: 1) animals that had a gradualdecline in health status over several days/weeks, and 2) animals thatappeared clinically to be tolerating the drug until shortly followingdosing on the day of early necropsy. For animals in the former category,there were clinical pathology changes suggestive of coagulopathy, acutephase response, severe dehydration, and/or liver toxicity, whereasanimals in the latter category were largely absent of clinical pathologychanges. Although there was a clinical history of rapid morbidity(within 1-3 minutes of dosing) and death (within 15 minutes of dosing)within category 2 animals was suggestive of drug instillation oraspiration into the lungs, the dose administration into the lungs couldnot be identified based on histopathology. The reason for the rapidonset of morbidity/mortality was unable to be determined but drugabsorption from the lungs could not be ruled out.

In the early death animals, drug-related histopathologic findings wereidentified in the lung, liver, spleen, mesenteric lymph node, andkidney. In the lung, gross and histologic findings consistent withpulmonary edema was identified in all dose groups and were generallymore severe than the findings, if any, in animals that survived to Day92 or 134.

In the surviving animals, there were CEP 28122-related clinical signspresent from all dose levels (20, 40, and 80/60 mg/kg) and included:emesis, decreased activity, hunched appearance, and watery feces. Ingeneral these clinical signs were not dose dependent (with the exceptionof watery feces). These clinical signs were not detected during therecovery phase of the study. There were CEP-28122-related increasedalanine aminotransferase (ALT) and aspartate aminotransferase (AST) inanimals dosed with 80/60 mg/kg suggestive of liver effects; however,these values had returned to baseline by the end of the recovery period.There were no definitive histological correlates in the liver.

CEP-28122-related changes in hematology parameters were limited to adecrease in lymphocytes in animals dosed with 80/60 mg/kg that began onDay 28 and persisted throughout the remaining time points during thedosing phase of the study. Lymphocyte counts had returned to baseline bythe end of the recovery period.

On Day 92, drug-related histopathologic findings were identified in thelung, liver, spleen, mesenteric lymph node, and kidney. Histologicfindings consistent with pulmonary edema and more chronic lung injurysimilar to those identified in early death animals were seen at Day 92and 134, were not dose-proportional, and were generally of lesserincidence and/or severity than the early death animals.Dose-proportional eosinophilic granularity within macrophages in theliver, spleen, and mesenteric lymph nodes was identified typically inanimals administered 40 and 80/60 mg/kg CEP-28122 with the inclusion ofminimal change in one animal administered 20 mg/kg CEP-28122. Thisfinding was associated with hepatocellular single-cell necrosis in theliver of one high-dose animal, but the increased incidence of the latterat Day 134 and its association with the eosinophilic granularity changesuggested an association with administration of the drug.Dose-proportional, brown, granular pigment within tubular epithelium ofthe kidney was identified in all drug-treatment groups, was minimal tomild as opposed to minimal to moderate in early death animals, and wasnot associated with degeneration/necrosis of tubular epithelium.

On Day 92, increases in organ weights and/or ratios related to theadministration of CEP-28122 were identified for the liver, lung, andkidney of male animals administered 40 and 80/60 mg/kg CEP-28122 (liverand kidney), and 20, 40 and 80/60 mg/kg CEP-28122 (lung). Theseincreases were correlated to eosinophilic granularity within Kupffercells and hepatocytes in the liver, pulmonary edema in the lung, andbrown, pigment accumulation in tubular epithelium in the kidney.

On Day 134, drug-related histopathologic findings were identified in thelung, liver, spleen, mesenteric and mandibular lymph nodes, and kidney.Histologic changes consistent with pulmonary edema were persistent withsimilar but slightly reduced severity in all dose groups compared toterminal necropsy animals, and were similarly non dose-proportional.Minimal change consistent with more chronic lung injury was present inone high dose animal.

Eosinophilic granularity within macrophages in the liver, spleen, andmesenteric and mandibular lymph nodes was identified typically inanimal(s) administered 40 and/or 80/60 mg/kg CEP-28122, and wasdose-proportional where more than one group was affected, with theexception of the spleen. This change in the liver was more severe ingeneral than that at Day 92. For the spleen, it was reduced in severitycompared to Day 92 for the 80/60 mg/kg group and of variable severity insingle occurrences in the 20 and 40 mg/kg groups. The degree of brown,granular pigmentation within areas of eosinophilic granularity withinthe liver and spleen was generally increased for these recovery groupscompared to earlier timepoints. Compared to Day 92, dose-proportional,brown, granular pigment within tubular epithelial cells of the kidneywas generally persistent but less severe in animals administered 40mg/kg CEP-28122, and persistent and more severe in animals administered80/60 mg/kg CEP-28122. Minimal degeneration/necrosis of tubularepithelium was identified in some animals administered 80/60 mg/kgCEP-28122 with the most severe pigment accumulation.

On Day 134, increases in organ weights and/or ratios related to theadministration of CEP-28122 were identified in females for the liver andkidney, were in animals administered 40 and/or 80/60 mg/kg CEP-28122,and were correlated similarly as in terminal necropsy animals.

Pulmonary edema was a significant concern in this study. In manyinstances the occurrence of pulmonary edema was asymptomatic prior torapid deterioration and mortality. Additionally, there was noperceptible cardiovascular component to the occurrence of the pulmonaryedema, thereby classifying this finding as noncardiogenic pulmonaryedema. The rapid onset and lack of a cardiovascular effect would make itvery difficult to diagnose or prevent the pulmonary edema in a clinicalsetting, rendering CEP-28122 unsafe for human use.

4-Week Oral Toxicity and Toxicokinetic Study of CEP-28122 in CynomolgusMonkeys with a 4-Week Recovery Period

Administration of CEP-28122 by oral gavage once daily for 4 weeks atdose levels of 3, 10, 20, and 40 mg/kg/day did not result in anymorbidity or mortality. There were no CEP-28122-related effects on foodconsumption, body weights, the lung (via auscultation), ocular, ECGs,blood pressure, heart rate, hematology, coagulation, urinalysis, urinechemistry, troponin I, or gross pathology observations at any dose levelevaluated.

CEP-28122-related histological effects occurred at dose levels ≧10mg/kg/day. These findings consisted of minimal or mild, multifocal,increase in vacuolated cells within the lung (presumptive alveolarepithelium) and minimal type II pneumocyte hyperplasia (20 mg/kg/daydose level only). The increased vacuolated cells correlated with anincreased lung weight and lung weight ratios from animals at dose levels≧10 mg/kg/day but did not correlate with any clinical observations orclinical lung findings via auscultation. Following a 4-week recoveryperiod, the increase in vacuolated cells within the lungs was stillpresent in animals at dose levels ≧20 mg/kg/day albeit with a lesserincidence and/or severity. Complete resolution of this finding did occurat the 10 mg/kg/day dose level. The type II pneumocyte hyperplasia wasnot detected from animals dosed with 20 mg/kg following the 4-weekrecovery period which also supported a minimal overall decrease inseverity.

Possible CEP-28122-related effects included post dose emesis in animalsat the 20 and 40 mg/kg dose levels and slight increases in triglycerides(40 mg/kg dose level only). These observations were not detected duringthe recovery period. Histopathological alterations in the retinal layersin one female dosed with 10 mg/kg/day on Day 29 was considered anuncertain CEP-28122-related effect. There were no correlates in theophthalmic exam.

This 4-week study at lower doses demonstrated that it would not bepossible to avoid lung toxicity with CEP-28122. Especially concerningwas the fact that lung damage was occurring without premonitory signs(auscultation). Based upon the occurrence and persistence of lungtoxicity in the 4- and 13-week monkey studies, it was concluded thatCEP-28122 was too dangerous for human use and development efforts wereterminated.

4-Week Oral Toxicity and Toxicokinetic Study of CEP-37440 inSprague-Dawley Rats with a 4-Week Recovery Period

No drug-related clinical observations were noted during the dosing orrecovery phase. Clinical observations appeared rather infrequently, weretransient, were with comparable incidences as controls, were associatedwith moribund animals, were associated with known gavage errors, oroccurred in animals whose deaths or sacrifices were not considereddrug-related; therefore, clinical observations were not considereddrug-related.

Males and females given 30 mg/kg/day gained less weight than controlsduring all intervals of the dosing phase. During Days 1 to 28 of thedosing phase, males gained 31% less than controls. During Days 1 to 28of the dosing phase, females gained 65% less than controls. Males andfemales given 30 mg/kg/day gained as much or more weight than controlsduring all intervals of the recovery phase. During Days 1 to 28 of therecovery phase, males gained 24% more than controls. During Days 1 to 28of the recovery phase, females gained 29% more than controls. Males andfemales given 30 mg/kg/day consumed less food than controls during allintervals of the dosing phase. These differences ranged from 4 to 10%less in males and from 12 to 24% less in females. No drug-relatedeffects on food consumption were noted during the recovery phase.Decreased mean terminal body weight was observed at the terminalsacrifice in males and females given 30 mg/kg/day (0.90× and 0.79×,respectively) and was statistically significant in females.

No drug-related ophthalmic findings were noted during the dosing orrecovery phase. No drug-related effects were observed in clinicalpathology test results up to 10 mg/kg/day. Several minor clinicalpathology effects were observed at 30 mg/kg/day that were minimal tomild in magnitude. None of these findings were considered adverse ortoxicologically important.

Drug hematology and coagulation findings at 30 mg/kg/day included thefollowing:

-   -   Mildly lower red cell mass (i.e., red blood cell count,        hemoglobin, and hematocrit) in females on Day 29 of the dosing        phase    -   Mildly lower absolute reticulocyte count in males on Days 15 and        29 of the dosing phase and in females on Day 15 of the dosing        phase    -   Mildly lower absolute neutrophils count in males on Days 15 and        29 of the dosing phase    -   Mildly lower absolute eosinophils count in females on Day 29 of        the dosing phase    -   Minimally higher fibrinogen in males on Days 15 and 29 of the        dosing phase

Platelet count appeared unaffected. Decreases in red cell mass, absolutereticulocyte, neutrophils, and eosinophil counts may have reflected mildbone marrow suppression/toxicity but correlative histopathology findingswere not observed in the bone marrow. These findings exhibitedreversibility at the end of the recovery phase. Higher mean corpuscularvolume in females given 30 mg/kg/day at the end of the recovery phasewere likely due to higher proportion of younger red cells that typicallyare larger in size.

Drug-related clinical chemistry findings at 30 mg/kg/day included thefollowing:

-   -   Mildly lower albumin in females on Day 29 of the dosing phase    -   Minimally higher globulin in females on Days 15 and 29 of the        dosing phase    -   Lower albumin-to-globulin ratio in females on Days 15 and 29 of        the dosing phase    -   Minimally higher cholesterol in males and females on Days 15 and        29 of the dosing phase    -   Mildly higher serum calcium concentration in males on Day 29 of        the dosing phase    -   Minimally lower serum chloride in males and females on Day 29 of        the dosing phase

Lower albumin and albumin-to-globulin ratio and higher globulin wereconsistent with inflammation and may have been associated with chronicinflammation of the thoracic cavity observed histologically. Highercholesterol may have been related to reduced food consumption and bodyweight gain observed in these animals. Lower serum chloride was likelydue to higher urine excretion. A mechanism was not known for highercalcium. No drug-related effect was observed in troponin I concentrationat any dose level. All clinical chemistry findings exhibitedreversibility at the end of the recovery phase.

Drug-related urine chemistry findings at 30 mg/kg/day included thefollowing:

-   -   Minimally higher urine chloride excretion in males and females        on Days 15 and 29 of the dosing phase    -   Minimally higher urine fractional clearance for chloride in        males and females on Days 15 and 29 of the dosing phase

Lower serum chloride was likely associated with higher total excretionand higher fractional clearance of chloride in the urine of animalsgiven 30 mg/kg/day. Correlative histopathology findings were notobserved in the kidney of these animals. All urine chemistry findingsexhibited reversibility at the end of the recovery phase. No apparenteffects were observed in urinalysis test results at any dose level.

Statistically significant or otherwise notable differences in otherclinical pathology test results were considered incidental because theywere usually of small magnitude, lacked a relationship to dose, or wereinconsistent over time and between sexes.

Seven unscheduled deaths occurred in toxicity animals during the dosingphase. None of the unscheduled deaths were attributed to the drug. Onecontrol male and one female given 30 mg/kg/day died soon after bloodcollection; these were considered accidental deaths due to the lack ofclinical or anatomical pathology findings suggesting a drug-relatedeffect. Three animals had macroscopic and/or microscopic findingsconsistent with gavage-related injuries, including one female given 1mg/kg/day sacrificed in moribund condition on Day 25, one female given 3mg/kg/day found dead on Day 9, and one female given 10 mg/kg/daysacrificed in moribund condition on Day 25 of the dosing phase. Thecause of death was not evident for one male given 30 mg/kg/day founddead on Day 10 and one female given 10 mg/kg/day sacrificed in moribundcondition on Day 15 of the dosing phase. All other dosing phase and allrecovery toxicity animals survived to their scheduled sacrifice.

At the terminal sacrifice, statistically significant increases in meanorgan weights (adjusted for terminal body weight) occurred in the liverof males given 30 mg/kg/day and prostate of males given 10 mg/kg/day.Because these changes lacked microscopic correlates, consistency betweensexes (liver only), and/or evidence of a dose response, they were notconsidered drug-related. Adjusted mean thymus weight was decreasedgreater than 10% and in a dose-dependent manner in males given 30mg/kg/day (0.86×) and females given 10 mg/kg/day (0.85×) or 30 mg/kg/day(0.79×). Lacking statistical significance or microscopic correlates, therelationship of thymus weight decreases to the drug in these groups isuncertain. All other organ weight changes at the terminal sacrifice andall organ weight changes at the recovery sacrifice were likely due tonormal biologic variation and were not considered a direct effect of thedrug.

Macroscopic findings of adhesions in the heart and adhesions or mass inthe lung were observed at the terminal sacrifice in females given 30mg/kg/day, and adhesions in the heart were observed at the recoverysacrifice in one female given 30 mg/kg/day. These macroscopic findingscorrelated with microscopic findings of fibrosis and/or chronicinflammation in the heart and lung and were considered drug-related. Allother macroscopic findings were considered spontaneous, incidental, orassociated with accidental death and were not attributed to the drug.

At the terminal sacrifice, microscopic findings consistent with chronicinflammation of the thoracic cavity were present on the serosal surfaceof the heart and lung and in perithymic connective tissue of femalesgiven 30 mg/kg/day. Chronic inflammation and/or fibrosis of theserosal/epicardial surface of the heart and pleural/subpleural surfaceof the lung was observed in 5/10 females given 30 mg/kg/day. Thefindings were multifocal to diffuse along the serosal surface of theheart and lung and varied from fibrous thickening with few inflammatorycells to more severe chronic inflammation. Chronic to chronic-activeinflammation was also observed in the loose fibrovascular connectivetissue surrounding the thymus in 4/10 females given 30 mg/kg/day. At therecovery sacrifice, chronic inflammation and/or fibrosis of the serosalsurface of the heart and lung were observed in ⅖ females given 30mg/kg/day. The findings at the recovery sacrifice were characterized bymultifocal to diffuse fibrous thickening of serosal surfaces with littleinflammation, suggesting partial resolution. Because findings of chronicinflammation in the thoracic cavity were observed at the scheduledsacrifices in a total of 7/15 females given 30 mg/kg/day and becausemacroscopic or microscopic evidence of another cause for these findingswas lacking, fibrosis and/or inflammation on serosal surfaces of theheart and lung and in perithymic fibroadipose tissue of females given 30mg/kg/day was considered most likely drug-related and may have resultedfrom serosal inflammation secondary to pleural and pericardialeffusions.

A small increase in incidence of alveolar macrophages in the lung wasobserved at the terminal sacrifice in females given 30 mg/kg/day and wasconsidered most likely drug-related. An increased incidence of alveolarmacrophages was not observed at the recovery sacrifice, suggesting thisfinding was reversible. All other microscopic findings at scheduled andunscheduled sacrifices were considered spontaneous, incidental, orassociated with accidental death and not attributable to the drug.

In a previously conducted 10-day dose range-finding study withCEP-37440, daily administration of the drug by gavage at 10, 30, or 60mg/kg/day to rats was well tolerated at 10 mg/kg/day. Drug-relatedadverse findings for body weight in combination with anatomic pathologyfindings in the bone marrow (hypocellularity); spleen, thymus, and lymphnode (decreased lymphocytes); and lung (increase in alveolarmacrophages) were observed in animals given ≧30 mg/kg/day. Drug-relatedchanges were not observed in animals given 10 mg/kg/day. In the currentstudy, adverse and drug-related findings were observed in males andfemales given 30 mg/kg/day. These findings included lower body weightgain in males and females, lower food consumption in males and females,and microscopic findings in females consistent with chronic inflammationof the thoracic cavity.

No drug-related effects were observed in clinical pathology test resultsup to 10 mg/kg/day. Several minor clinical pathology effects wereobserved at 30 mg/kg/day that were minimal to mild in magnitude. None ofthese findings were considered adverse, toxicologically important, ordefinitively correlated with other adverse in life or anatomic pathologyfindings.

Decreases in red cell mass, absolute reticulocyte, neutrophil, andeosinophil counts may have reflected mild bone marrowsuppression/toxicity, but correlative histopathology findings were notobserved in the bone marrow. These findings exhibited reversibility atthe end of the recovery phase. Higher mean corpuscular volume at the endof the recovery phase in females given 30 mg/kg/day was likely due tohigher proportion of younger red cells that typically are larger insize.

Lower albumin and albumin-to-globulin ratio and higher globulin wereconsistent with inflammation and may have been associated with chronicinflammation of the thoracic cavity observed histologically. Highercholesterol may have been related to reduced food consumption and bodyweight gain observed in these animals. Lower serum chloride was likelydue to higher urine excretion. A mechanism was not known for highercalcium. No drug-related effect was observed in troponin I concentrationat any dose level. All clinical chemistry findings exhibitedreversibility at the end of the recovery phase.

Lower serum chloride was likely associated with higher total excretionand higher fractional clearance of chloride in the urine of animalsgiven 30 mg/kg/day. Correlative histopathology findings were notobserved in the kidney of these animals, and all urine chemistryfindings exhibited reversibility at the end of the recovery phase.

In conclusion, oral CEP-37440 administration to rats at doses ≦10mg/kg/day for 28 days was clinically well tolerated. Microscopicfindings consistent with chronic inflammation of the thoracic cavitywere present in females given 30 mg/kg/day. Decreases in body weightchange and food consumption were present in males and females given 30mg/kg/day. Based on these findings, the no observed adverse effect level(NOAEL) in this study was 10 mg/kg/day.

4-Week Oral Toxicity and Toxicokinetic Study of CEP-37440 in CynomolgusMonkeys with a 4-Week Recovery Period

All animals survived until their scheduled sacrifice.

No drug-related clinical observations were noted during the dosing orrecovery phase. Clinical observations appeared rather infrequently, weretransient, or were with comparable incidences as controls; therefore,they were not considered drug-related.

There was no drug-related effects on body weight or body weight gainnoted during the dosing or recovery phase. There were no drug-relatedabnormal ophthalmic findings noted. Additionally, there were nodrug-related observations noted during blood pressure measurements.

No drug-related changes in PR interval, QRS duration, QT interval,corrected QT (QTc) interval, RR interval, or heart rate were observed onDays 3 and 27 of the dosing phase or Day 28 of the recovery phase inanimals given 2.5, 7.5, or 20.0 mg CEP-37440/kg of body weight/day(mg/kg/day). No rhythm abnormalities or qualitative ECG changesattributable to CEP-37440 were observed during qualitative assessment ofthe ECGs.

CEP-37440 administration up to 20.0 mg/kg/day had no effect on clinicalpathology test results at the end of dosing or recovery phase.

A few individual animals had clinical pathology findings consistent withinflammation (including slight to notable increases in white blood celland absolute neutrophils counts, fibrinogen, and C-reactive protein)during the dosing phase, but these animals were usually scattered acrossall groups, including the control. In that regard, these findings wereconsidered unrelated to the drug because they lacked a dose-relatedpattern, were often inconsistent over time (especially for leukocytecounts), and involved some control animals.

There were no statistically significant changes in terminal body ororgan weights at the terminal sacrifice. All organ weight changespresent at the scheduled terminal or recovery sacrifice were attributedto normal biologic variation and considered incidental and unrelated tothe drug.

No clear, drug-related macroscopic findings were present at the terminalor recovery sacrifice. At the terminal sacrifice, all (3/3) males given7.5 mg/kg/day and ⅔ females given 20.0 mg/kg/day exhibited single tofew, red to dark red foci on the mucosal surface of the stomach. Theserosa of the stomach in one male given 20.0 mg/kg/day exhibited a fewred areas. Microscopic correlates were focal hemorrhage within themucosa and/or submucosa/tunica muscularis, with or without the presenceof smooth muscle degeneration within the tunica muscularis. The presenceof similar findings in one control female suggests these findings areincidental and unrelated to the drug.

No clear, drug-related microscopic findings were present at the terminalor recovery sacrifice. Macroscopic findings in the mucosa or serosa ofthe stomach in some animals given CEP-37440 generally correlated withmicroscopic findings of focal hemorrhage within the mucosa and/orsubmucosa/tunica muscularis, with or without the presence of smoothmuscle degeneration within the tunica muscularis. However, theassociation of macroscopic and microscopic findings to CEP-37440administration in the current study is uncertain given the presence ofsimilar and more severe microscopic findings in the stomach of a femalegiven control article and the fact that gastric smooth muscledegeneration is a known background finding in cynomolgus monkeys. Othermicroscopic findings were present in the stomach of one or more animals,but their association to the drug is uncertain because of their lowseverity and/or incidence, lack of a clear dose-response, or concurrentpresence in a control animal.

All remaining microscopic findings at the terminal or recoverysacrifice, including minimal, focal infiltrates of alveolar macrophagesin the lung of two females given 20.0 mg/kg/day, were attributed tonormal biologic variation and considered incidental.

In conclusion, oral CEP-37440 administration to cynomolgus monkeys atdoses up to 20 mg/kg/day for 28 consecutive days was well tolerated, andno drug-related effects were observed at any dose level. Significantly,there was no evidence whatsoever of pulmonary edema, and CEP-37440 wasdetermined to be safe for use in human trials. The superior safetyprofile of CEP-37440 in comparison to CEP-28122, and in particular thelack of lung toxicity, was surprising and unexpected.

Anti-Tumor Efficacy in NPM-ALK Positive Sup-M2 and Karpas-299 ALCL TumorXenograft Models in Mice

No significant anti-tumor activity is observed following 12 daytreatment with CEP-37440 at 10 mg/kg or lower, bid; partial tumorregression is observed following 12 day treatment with CEP-37440 at 30mg/kg, qd; complete or near complete tumor regression is observedfollowing 12 day treatment with CEP-37440 at 30 mg/kg bid or 55 mg/kg,qd (FIG. 4). Administration of CEP-37440 is well tolerated with no overttoxicity and no significant compound-related body weight loss of mice atall dosing regimens (FIG. 5). Dose-related levels of CEP-37440 are foundin plasma and tumor lysates collected at 2 h post final dosing (FIG. 6).Note that no CEP-37440 is observable in tumors at the 30 mg/kg, bid and55 mg/kg, qd dosing levels because those animals had no tumors—completetumor regression. CEP-37440 levels are approximately 2-3-fold higher inplasma and more than 10-fold higher in tumors than the levels at 2 hpost single oral dose in PK/PD studies, suggesting some compoundaccumulation in plasma and tumors with bid or qd oral dosing regimes at10 and 30 mg/kg.

In Karpas-299 tumor xenografts, significant anti-tumor activity isobserved at 30 mg/kg qd, and complete or near complete tumor regressionis observed following 12 day treatment at 30 mg/kg bid or 55 mg/kg, qd(FIG. 7). CEP-37440 administration is well tolerated with no overttoxicity and no significant body weight loss at the dosing regimens(FIG. 8). Dose-related levels of CEP-37440 are observed in plasma andtumor lysates collected at 2 h post final dosing (FIG. 9). Note that noCEP-37440 is observable in tumors at the 50 mg/kg, qd dosing levelbecause those animals had no tumors—complete tumor regression.

Anti-Tumor Activity in EML4-ALK Positive (NCI-H2228 and NCI-H3122) NSCLCTumor Xenografts in Mice with Oral Dosing

For the NCI-H2228 tumor xenograft models, treatment with CEP-37440 (HClsalt) at 30 mg/kg, qd and bid and 55 mg/kg, qd po for 12 days results intumor regressions (FIG. 10). For the NCI-H3122 tumor xenograft models,treatment with CEP-37440 (HCl salt) at 30 mg/kg, bid or 55 mg/kg, qd, pofor 12 days results in tumor stasis and partial regressions (FIG. 11).The improved anti-tumor efficacy observed in NCI-H2228 tumor xenograftsis likely due to the higher tumor distribution of CEP-37440 (FIGS. 12and 13). Treatment in these tumor-bearing mice was well tolerated withno overt toxicity or compound-related weight loss (FIGS. 14 and 15),except at 30 mg/kg, bid dose in NCI-H3122 tumor-bearing mice (FIG. 15).

Four weeks of additional treatment of the NCI-H2228 tumor-bearing miceat 55 mg/kg qd po provides sustained complete tumor regression in 100%of the animals (FIG. 16). The additional treatment is well toleratedwith no overt toxicity and no significant body weight loss (FIG. 17).Sustained tumor regressions are observed in 100% of the mice for 40 daysfollowing cessation of treatment, with no tumor re-emergence in anymouse (FIG. 16).

This is important because it suggests that the tumors are completelyeradicated and that the mice are effectively “cured” after approximately6 weeks of treatment with CEP-37440.

Anti-Tumor Efficacy Studies in Human Tumor Xenografts ofHormone-Independent Prostate Carcinoma, NSCL Carcinoma and HNSCCarcinoma

In established FAK-positive PC-3 prostate tumor xenografts,administration of CEP-37440 over a 36 day period results in a 55% tumorgrowth inhibition (TGI) and 10% incidence of complete tumor regressions,a profile similar to that of an equivalent dose of PF-562271 in thismodel (69% TGI and 25% incidence of partial tumor regressions) (FIG.18). All dosing regimens are well tolerated with no overt toxicity orsignificant body weight loss observed.

Non-Small Cell Lung (NSCL) Carcinoma

CEP-37440 demonstrates dose-related anti-tumor efficacy, with 80% TGIand a 60% incidence of tumor regressions (30% complete and 30% partial)at 55 mg/kg bid and significant efficacy (60% TGI and evidence forpartial tumor regression) at 30 mg/kg bid by day 28 of the study (FIG.19). Significant anti-tumor efficacy (66% TGI) is observed withPF-562271 at 55 mg/kg bid, but modest tumor growth rebound was observedbeginning at day 23. Administration of both CEP-37440 and PF-562271 arewell tolerated, with no overt toxicity or significant body weight lossobserved (FIG. 20). Of note, the significant efficacy achieved withCEP-37440 is not the result of inhibiting EGF-R phosphorylation(activation) in this EML4-ALK negative tumor xenograft model.

Head and Neck Squamous Cell Carcinoma (HNSCC)

In SCID mice bearing established Detroit 562 HNSCC xenografts, CEP-37440and PF-562271 demonstrated a clear tumor pharmacodynamic effect forinhibition of FAK activation with no effect on total FAK expressionlevels (FIG. 21). Over a 28-day period, CEP-37440 and PF-562271 resultin tumor stasis and a 20% incidence (CEP-37440, 30 mg/kg bid) and 30%incidence (CEP-37440 and PF-562271, 55 mg/kg bid) of partial tumorregressions. The magnitude of efficacy observed with CEP-37440 (bothdoses) is comparable to that observed with 55 mg/kg bid of PF-562271(Roberts et al., 2008). The dosing regimens are well-tolerated with nomorbidity or mortality observed.

These studies are important because they show that CEP-37440demonstrates significant FAK pharmacodynamic inhibition andALK-independent anti-tumor efficacy in established xenogaft models ofhormone-independent prostate carcinoma, NSCL carcinoma, andHNSCC—including objective tumor responses.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application and thescope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of treating an ALK- or FAK-mediatedproliferative disorder in a subject, comprising administering to thesubject a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of Formula (I):

or a salt thereof and at least one excipient.
 2. The method of claim 1,wherein the salt of the compound of Formula (I) comprises an acidaddition salt.
 3. The method of claim 1, wherein the salt of thecompound of Formula (I) comprises an tribenzenesulfonate salt.
 4. Themethod of claim 3, wherein the tribenzenesulfonate salt of Formula (I)has an XRPD pattern comprising one or more peaks selected from 7.62±0.2degrees 2θ, 13.11±0.2 degrees 2θ, 13.76±0.2 degrees 2θ, and 14.05±0.2degrees 2θ.
 5. The method of claim 3, wherein the tribenzenesulfonatesalt of Formula (I) has an XRPD pattern comprising one or more peaksselected from 6.85±0.2 degrees 2θ, 7.62±0.2 degrees 2θ, 8.01±0.2 degrees2θ, 13.11±0.2 degrees 2θ, 13.76±0.2 degrees 2θ, 14.05±0.2 degrees 2θ,and 14.60±0.2 degrees 2θ.
 6. The method of claim 3, wherein thetribenzenesulfonate salt of Formula (I) has an XRPD pattern comprisingone or more peaks selected from 7.62±0.2 degrees 2θ, 13.11±0.2 degrees2θ, 13.76±0.2 degrees 2θ, 14.05±0.2 degrees 2θ, 17.10±0.2 degrees 2θ,17.86±0.2 degrees 2θ, and 18.10±0.2 degrees 2θ.
 7. The method of claim1, wherein the salt of the compound of Formula (I) comprises atrihydrochloride dihydrate salt.
 8. The method of claim 7, wherein thetrihydrochloride dehydrate salt of Formula (I) has an XRPD patterncomprising one or more peaks selected from 5.42±0.2 degrees 2θ, 8.86±0.2degrees 2θ, 14.06±0.2 degrees 2θ, 17.52±0.2 degrees 2θ and 18.51±0.2degrees 2θ.
 9. The method of claim 7, wherein the trihydrochloridedehydrate salt of Formula (I) has an XRPD pattern comprising one or morepeaks selected from 5.42±0.2 degrees 2θ, 5.91±0.2 degrees 2θ, 8.86±0.2degrees 2θ, 10.80±0.2 degrees 2θ, 11.79±0.2 degrees 2θ, 14.06±0.2degrees 2θ, 14.72±0.2 degrees 2θ, 17.02±0.2 degrees 2θ, 17.52±0.2degrees 2θ and 18.51±0.2 degrees 2θ.
 10. The method of claim 1, whereinsaid pharmaceutical composition comprises a liquid formulation.
 11. Themethod of claim 10, wherein the liquid formulation is selected from thegroup consisting of a solution, a suspension, and an emulsion.
 12. Themethod of claim 1, wherein said pharmaceutical composition comprises asolid formulation.
 13. The method of claim 12, wherein said solidformulation is selected from the group consisting of a powder, a tablet,a capsule, a cachet, a suppository, and a dispersible granule.
 14. Themethod of claim 12, wherein said solid formulation is a tablet or acapsule.
 15. The method of claim 1, wherein the ALK- or FAK-mediatedproliferative disorder is selected from the group consisting ofanaplastic large cell lymphoma (ALCL), non-small cell lung cancer(NSCLC), neuroblastoma, glioblastoma, prostate cancer, squamous cellcarcinoma (SCC), and breast cancer.
 16. The method of claim 14, whereinthe ALK- or FAK-mediated proliferative disorder is selected from thegroup consisting of ALK-positive ALCL, EML4-ALK-positive NSCLC,neuroblastoma, glioblastoma, androgen-independent prostate cancers,breast cancers, and head and neck squamous cell carcinomas (HNSCCs). 17.The method of claim 14, wherein the ALK- or FAK-mediated proliferativedisorder is selected from the group consisting of ALK-positive ALCL,EML4-ALK-positive NSCLC, neuroblastoma, androgen-independent prostatecancers, breast cancers, and HNSCCs.
 18. The method of claim 14, whereinthe ALK- or FAK-mediated proliferative disorder is selected from thegroup consisting of ALK-positive ALCL, EML4-ALK-positive NSCLC,neuroblastoma, and glioblastoma.
 19. The method of claim 14, wherein theALK- or FAK-mediated proliferative disorder is selected from the groupconsisting of ALK-positive ALCL, EML4-ALK-positive NSCLC, andneuroblastoma.
 20. The method of claim 14, wherein the ALK- orFAK-mediated proliferative disorder is selected from the groupconsisting of ALK-positive ALCL and EML4-ALK-positive NSCLC.
 21. Themethod of claim 14, wherein the ALK- or FAK-mediated proliferativedisorder is selected from the group consisting of androgen-independentprostate cancers, breast cancers, and HNSCCs.